new tools to study transgene expression in chloroplasts ...jul 27, 2016 · 3 52 introduction 53...

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New tools to study transgene expression in chloroplasts 1

Codon-optimization to enhance expression yields insights into chloroplast translation 2

Kwang-Chul Kwon1, Hui-Ting Chan1, Ileana R. León2, Rosalind Williams-Carrier3, Alice 3

Barkan3, and Henry Daniell1* 4

1Department of Biochemistry, School of Dental Medicine, University of Pennsylvania 5

Philadelphia, PA 19104-6030, USA; 2 Global Research, Novo Nordisk A/S, Måløv, DK-2760, 6

Denmark; 3Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229 7 8

K.C.K. organized codon tables, created and characterized transplastomic plants, interpreted and 9

wrote sections of this manuscript (Fig. 1-4, Fig. S1-S4 and supplemental data set). H.T.C created 10

and characterized transplastomic plantsand contributed data in Fig. 2D, 4B. I.R.L. performed MS 11

and PRM analyses, interpreted data and wrote this manuscript section (Fig. 5-6, Fig. S4-S5 and 12

supplemental data set). R.W-C. contributed ribosome profiling data analyses (Fig.7). A.B. 13

interpreted ribosome profiling data, wrote this manuscript section. H.D. conceived and designed 14

this project, analyzed and interpreted data and wrote and revised several sections and versions of 15

this manuscript. 16

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SUMMARY 18

Eukaryotic genes coding for biopharmaceutical proteins expressed in chloroplasts using different codons but 19

identical regulatory sequences shed light on key factors that limit or enhance protein synthesis. 20

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*Corresponding Author 22

Henry Daniell, Ph. D., Professor and Director of Translational Research, University of 23

Pennsylvania, Philadelphia; Email: [email protected]; Tel: 215-746-2563; 24

Fax: 215-898-3695 25

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Plant Physiology Preview. Published on July 27, 2016, as DOI:10.1104/pp.16.00981

Copyright 2016 by the American Society of Plant Biologists

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Abstract 29

Codon optimization based on psbA genes from 133 plant species eliminated 105 (human 30

clotting factor VIII heavy chain, FVIII HC) and 59 (polio viral capsid protein 1, VP1) rare 31

codons; replacement with only the most highly preferred codons decreased transgene expression 32

(77-111-fold) when compared to codon usage hierarchy of the psbA genes. Targeted proteomic 33

quantification by parallel reaction monitoring (PRM) analysis showed 4.9-7.1 or 22.5-28.1-fold 34

increase in FVIII or VP1 codon optimized genes when normalized with stable isotope-labeled 35

standard (SIS) peptides (or house-keeping protein peptides) but quantitation using western blots 36

showed 6.3-8.0 or 91-125-fold increase of transgene expression from the same batch of materials 37

due to limitations in quantitative protein transfer, denaturation, solubility or stability. PRM, 38

validated here for the first time for in planta quantitation of biopharmaceuticals is especially 39

useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots 40

confirmed that the increase of codon-optimized protein synthesis is at the translational level 41

rather than any impact on transcript abundance. Ribosome footprints did not increase 42

proportionately with VP1 translation or even decreased after FVIII codon optimization but is 43

useful in diagnosing additional rate limiting steps. A major ribosome pause at CTC leucine 44

codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls 45

observed at clusters of serine codons in the codon-optimized VP1 gene provides opportunity for 46

further optimization. In addition to increasing our understanding of chloroplast translation, these 47

new tools should help to advance this concept towards human clinical studies. 48

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3

Introduction 52

Heterologous gene expression has facilitated understanding of DNA replication, 53

recombination, transcription, translation and protein import in chloroplasts. Expression of 54

precursor proteins via the chloroplast genome demonstrated that cleavage of transit peptides 55

takes place in the stroma and not in the chloroplast envelope (Daniell et al., 1998). Most 56

importantly, the role of nuclear-encoded cytosolic proteins that bind to regulatory sequences and 57

their species specificity was demonstrated using transgenes expressed in chloroplasts (Ruhlman 58

et al., 2010). When the lettuce psbA regulatory sequence was used to drive transgene expression 59

in tobacco chloroplasts, there was >90% reduction in accumulation of foreign proteins. This 60

underscores the importance of species-specificity of chloroplast regulatory sequences. Likewise, 61

details of homologous recombination process, and the deletion of mismatched nucleotides were 62

evident using heterologous flanking sequences (Ruhlman et al., 2010). Translation of native 63

polycistrons without need for processing to monocistrons has been demonstrated (Barkan, 1988; 64

Zoschke et al., 2015) but similarity of this process using heterologous polycistrons engineered 65

via the chloroplast genome offered even more direct evidence for this process (De Cosa et al., 66

2001; Quesada-Vargas et al., 2005). Insertion of replication origins into chloroplast vectors 67

offered further insight into minimal sequences required to study this process (Daniell et al., 68

1990). Therefore, in this study we use transgenes, chloroplast genome sequences and cutting-69

edge tools to understand the process of translation in chloroplasts. 70

Each plant cell contains up to 10,000 copies of the chloroplast genome. Therefore, 71

transgenes inserted into chloroplast genomes are expressed at high levels - up to 70% of total leaf 72

protein (De Cosa et al., 2001; Ruhlman et al., 2010). A wide range of proteins, from very small 73

antimicrobial peptides (Lee et al., 2011) or hormones (Boyhan and Daniell, 2011; Kwon et al., 74



4

2013) to very large proteins encoded by bacterial, viral, fungal, animal and human genes have 75

been successfully expressed in plant chloroplasts (DeGray et al., 2001; Daniell et al., 2009; 76

Verma et al., 2010; Shenoy et al., 2014; Sherman et al., 2014; Shil et al., 2014). Most 77

importantly, expressed proteins are highly stable when lyophilized plant cells are stored at 78

ambient temperature (Kwon et al., 2013; Lakshmi et al., 2013; Kohli et al., 2014; Jin and Daniell, 79

2015). Therefore, oral delivery of proinsulin or exendin-4 reduced blood sugar levels similar to 80

injected proteins (Boyhan and Daniell 2011; Kwon et al., 2013). Oral delivery of angiotensin and 81

angiotensin converting enzyme 2 (ACE2) expressed in chloroplasts reversed or prevented 82

pulmonary hypertension by shifting the renin-angiotensin system (RAS) to its protective axis, 83

resulting in a decrease in fibrosis, improvement in cardiopulmonary structure and function, and 84

restoration of right heart function (Shenoy et al., 2014). Furthermore, ocular inflammation 85

caused by decreased activity of the protective axis of RAS was significantly improved (Shil et al., 86

2014). Likewise, oral delivery of myelin basic protein reduced Aβ plaques in advanced mouse 87

and human Alzheimer’s brains (Kohli et al., 2014). Delivery of coagulation factors to hemophilic 88

mice induced oral tolerance and suppressed inhibitor formation and anaphylaxis (Verma et al., 89

2010; Sherman et al., 2014; Wang et al., 2015a). Aforementioned examples illustrate the 90

significance of this novel, cost-effective protein drug delivery concept. 91

However, a major limitation in clinical translation of human therapeutic proteins in 92

chloroplasts is their low-level expression. Prokaryotic or shorter human genes are highly 93

expressed in chloroplasts (De Cosa et al., 2001; Ruhlman et al., 2010; Daniell et al., 2009; Arlen 94

et al., 2007). However, expression of larger human proteins is a major challenge. For example, 95

cholera non-toxic B subunit (CNTB)-fused native human blood clotting factor VIII heavy chain 96

(FVIII HC, 86.4 kDa) or angiotensin converting enzyme 2 (ACE2, 92.5 kDa) were expressed at 97



5

very low levels (Shenoy et al., 2014). Likewise, expression of viral vaccine antigens is quite 98

unpredictable with high, moderate or extremely low expression levels (Birch-Machin et al., 2004; 99

Inka Borchers et al., 2012). Lenzi et al., 2008; Waheed et al., 2011a; Waheed et al., 2011b; 100

Hassan et al., 2014). Furthermore, viral antigens are highly unstable with expression observed in 101

youngest leaves but not in mature leaves (McCabe et al., 2008). It is well known that high doses 102

of vaccine antigens stimulate high-level immunity and confer greater protection against 103

pathogens; therefore, higher-level expression in chloroplasts is a key requirement for vaccine 104

development (Chan and Daniell, 2015; Chan et al 2016). 105

Such challenges in transgene expression have been addressed by the use of optimal 106

regulatory sequences (promoters, 5ʹ- and 3ʹ-UTRs), especially species-specific endogenous 107

elements (Ruhlman et al., 2010). In vitro assays of inserted genes with several synonymous 108

codons show that translation efficiency does not always correlate with codon usage in plastid 109

mRNAs (Nakamura and Sugiura, 2007) but they have been used in several codon optimization 110

studies (Ye et al., 2001; Lutz et al., 2001; Franklin et al., 2002; Lenzi et al., 2008; Madesis et al., 111

2010; Jabeen at al., 2010; Gisby et al., 2011; Wang et al., 2015b; Boehm et al., 2016; Nakamura 112

et al., 2016). While some studies achieved significant increases in expression (75-80 fold) after 113

codon optimization (Gisby et al 2011; Franklin et al 2002), other studies observed negligible 114

enhancement (Lenzi et al., 2008; Nakamura et al., 2016; Ye et al., 2001; Lenzi et al., 2008; 115

Wang et al., 2015b; Daniell et al 2009). However, translation initiation and elongation efficiency 116

of codon optimized sequences were enhanced when chloroplast gene N-terminal sequences were 117

inserted downstream of 5ʹ- UTRs (Ye et al., 2001; Lenzi 2008). In a recent study (Nakamura et 118

al., 2016), the importance of compatibility between the psbA 5ʹ-UTR and its 5ʹ coding sequence 119

was shown using codon-optimized heterologous genes. Aforementioned codon optimization 120



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studies used only smaller eukaryotic coding sequences (<30 kDa) but there is a great need to 121

express larger human genes (e.g. Human blood clotting factor VIII >200kDa) that would require 122

optimization of not only codons but also compatibility with regulatory sequences for optimal 123

translation initiation, elongation and greater understanding of tRNAs encoded by the chloroplast 124

genome or imported from the cytosol. However, no systematic study has been done to utilize the 125

extensive knowledge gathered by sequencing several hundred chloroplast genomes to understand 126

codon usage and frequency of highly expressed chloroplast genes. 127

Another major challenge is the lack of reliable methods to quantify insoluble proteins; the 128

only reliable method (ELISA) cannot be used due to aggregation or formation of multimeric 129

structures that are required for oral drug delivery. Although the FDA accepts ELISA for 130

quantitation of purified protein drugs, it is not suitable for quantifying protein drugs from impure 131

extracts due to cross-reacting proteins, autoantibodies (Kim and You, 2013) or for quantitation of 132

insoluble, multimeric or membrane proteins. Similarly, immunoblots used for quantitation also 133

have several limitations, i.e. aggregation of proteins at high protein concentrations trapped in 134

wells, alteration of mobility by incomplete solubilization or secondary structures, saturation of 135

antibody binding sites and inefficient transfer of large proteins to membranes and variable 136

quantitation due to short or long exposure to films. However, peptide centric quantitation 137

strategies (e.g. targeted mass spectrometry quantitation by parallel reaction monitoring, PRM) 138

can overcome most of the limitations mentioned above. In the preparation of protein samples for 139

PRM, strong denaturing and reducing conditions are used (e.g. higher concentrations of SDS and 140

DTT) in combination with optimal enzymatic proteolysis conditions (e.g. sodium-deoxycholate, 141

León et al 2013), especially suitable for insoluble, multimeric or membrane proteins (Savas, et al. 142

2011). Moreover, PRM can be used for relative and absolute protein quantitation of target 143



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proteins present in highly complex protein background based on its high specificity and 144

sensitivity (Domon and Aebersold, 2010; Gallien et al., 2012; Picotti and Aebersold, 2012). In 145

addition, PRM offers high specificity and multiplexing characteristics, which allow for specific 146

monitoring of up to several hundred peptides in a single analysis (Gallien et al., 2012). 147

Determination of protein drug dose in planta, especially of insoluble proteins without 148

purification is an unexplored area of research and we investigate this concept for the first time to 149

quantify recombinant protein drugs made in chloroplasts. 150

This study explores heterologous gene expression utilizing chloroplast genome sequences, 151

ribosome profiling and targeted mass spectrometry (PRM) to enhance our understanding of 152

translation of foreign genes in chloroplasts. We developed a codon optimizer program based on 153

the analysis of psbA genes from 133 plant species to compare translational efficiencies of native 154

and codon-optimized genes driven by identical regulatory sequences. PRM using peptides 155

selected from the N- or C-terminus were used to study complete or incomplete synthesis of 156

proteins and to validate this approach to quantify the dosage of protein drugs made in plant cells 157

when compared with current methods. The codon optimizer program was evaluated in 158

chloroplasts from two different species to identify any species specificity. Ribosome profile was 159

evaluated for its suitability to diagnose limiting steps in transgene expression. These 160

observations provide new insight into limitations in translation of heterologous genes and 161

approaches to address this in future studies. 162

Results 163

Codon optimization of human/viral transgenes 164

Differences in codon usage by chloroplasts frequently decrease translation. We observed 165

that plants expressing native sequences of the blood clotting factor VIII heavy chain (FVIII HC) 166



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or capsid protein 1 (VP1) from polio virus showed very low levels of expression, <0.05 % for 167

FVIII and ~0.1% for VP1 (see data shown below). The psbA gene is one among the most highly 168

expressed genes in chloroplasts and the translation efficiency of the psbA gene is >200 times 169

higher than the rbcL gene (Eibl et al., 1999). The 5ʹ-UTR of psbA also showed the highest 170

translation activity in vitro among eleven 5ʹ-UTRs investigated (Yukawa et al. 2007). Therefore, 171

among 140 transgenes expressed in chloroplasts, >75% use the psbA regulatory sequences 172

(Daniell et al., 2016). Most importantly, compatibility between the 5ʹ-UTR of psbA and its 173

coding region is important for efficient translation initiation (Nakamura et al., 2016). For these 174

reasons, a new codon optimization program was developed using codon usage of the psbA genes 175

from 133 sequenced chloroplast genomes (Fig 1A). We first investigated expression of synthetic 176

genes using only the most highly preferred codon for each amino acid, which is referred to as the 177

“old” algorithm in this study. When this resulted in even lower levels of expression than the 178

native gene (see data presented below), a “new” codon optimizer algorithm was developed using 179

codon usage hierarchy observed among sequenced psbA genes. Therefore, most of the rare 180

codons in heterologous genes were modified based on codons with >5% frequency of use in the 181

psbA genes. Synonymous codons for each amino acid were ranked according to their frequency 182

of use (Fig. 1B). 183

In this study, native sequences for FVIII HC (2262 bp) and VP1 (906) were codon-184

optimized using the “old” or “new” algorithms and synthesized. After codon optimization, the 185

AT content of FVIII HC increased slightly from 56% to 62%, and 406 codons out of 754 amino 186

acids were optimized. For the VP1 sequence from Sabin 1 polio virus strain, the 906-bp long 187

native sequence was codon-optimized, which slightly increased the AT content from 52.0% to 188

59.0%, and 187 codons out of 302 amino acids were optimized. However, the CNTB coding 189



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sequence was not codon-optimized because of its prokaryotic origin and high AT content 190

(65.4%). Most importantly, the expression level of CNTB (native sequence) fused with 191

proinsulin in tobacco chloroplasts reached up to 72% of total leaf protein (Ruhlman et al., 2010) 192

and 53% of total leaf protein in lettuce chloroplasts (Boyhan and Daniell, 2011), indicating that 193

there is no limitation on translation of the CNTB coding sequence in chloroplasts. All sequences, 194

including native and codon-optimized synthetic genes (new and old algorithms) are shown in 195

Supplemental Fig. 1S.; rare codons in native genes are shown in red font and modified codons 196

are highlighted in yellow in Supplemental Fig. 2S. 197

When the psbA-based codon table is compared with total chloroplast codon usage tables, 198

which are generated based on all chloroplast genes of Lactuca sativa (57,528 codons from 189 199

coding sequences) or Nicotiana tabacum (34,756 codons from 137 coding sequences) 200

(Nakamura et al., 2000), there was no significant difference in AT content of coding sequences; 201

it varied between 59.59% and 61.76%. However, there are striking differences between psbA-202

based and total chloroplast gene-based codon tables when individual codons are compared. 203

Native FVIII HC used CTC leucine codon 11 times but codon-optimized (new algorithm) HC 204

eliminated all CTC codons. However, if total chloroplast codon table is used, codon-optimized 205

HC would still use 5 CTC codons. As seen in ribosome profiles, discussed below, tandem repeat 206

of CTC-CTC in the native FVIII HC sequence resulted in major stalling sites which were 207

completely eliminated by psbA based codon optimization (new algorithm). Likewise, another 208

rare codon TCA (serine) is used 16 times in the FVIIIHC and 7 times in VP1 coding sequences. 209

However, the TCA rare codon was completely eliminated in both genes after codon optimization 210

using the new algorithm. However, if the total codon table is used for codon optimization, FVIII 211

HC and VP1 would still contain 12 or 5 TCA codons. Collectively, the new codon-optimization 212



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algorithm eliminated 105 and 59 rare codons from FVIII HC and VP1, respectively, resulting in 213

enhanced expression of both genes. However, if the total codon table is used, there will be 75 214

and 35 rare codons in codon-optimized FVIII HC and VP1 coding sequences, respectively. All 215

thirteen codons [GCG (Ala), GGG (Gly), CTG (Leu), CTC (Leu), CCG (Pro), CCC (Pro), AGG 216

(Arg), CGG (Arg), TCA (Ser), TCG (Ser), ACG (Tyr), GTC (Val), CTG (Val)] rarely used in the 217

psbA gene were eliminated using our codon-optimized table (new algorithm). More detailed 218

information on codon distribution between different codon tables is included in Supplemental 219

data (Fig. S3). 220

Synthetic gene cassettes were inserted into the chloroplast transformation vector, pLSLF 221

for lettuce or pLD-utr for tobacco (Fig. 2A). Native and synthetic genes were fused to the native 222

CTNB sequence, which is used for efficient transmucosal delivery of fused proteins via 223

monosialotetrahexosylganglioside receptors (GM1) present on intestinal epithelial cells. To 224

eliminate possible steric hindrance caused by the fusion of two proteins and facilitate the release 225

of tethered proteins into circulation after internalization, nucleotide sequences for a hinge (Gly-226

Pro-Gly-Pro) and a furin cleavage site (Arg-Arg-Lys-Arg) were engineered between CNTB and 227

fused proteins. Fusion genes were placed under identical psbA promoter, 5’-UTR and 3’-UTR 228

regulatory sequences for specific evaluation of codon optimization (Fig. 2A). To select 229

transformants, the aminoglycoside-3″-adenylyl-transferase gene (aadA) was driven by the 230

ribosomal RNA promoter (Prrn) to confer resistance to spectinomycin in transformed cells. 231

Expression cassettes were flanked by sequences for isoleucyl-tRNA synthetase (trnI) and alanyl-232

tRNA synthetase (trnA), which are identical to endogenous chloroplast genome sequences, 233

leading to efficient double homologous recombination and optimal processing of introns with 234

flanking sequences (Fig. 2A). 235



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Transformation vectors containing the native or synthetic sequences for FVIII HC and 236

VP1 sequences were used to create transplastomic lettuce or tobacco plants. To confirm 237

homoplasmy, Southern blot analysis was performed on four independent lettuce and tobacco 238

lines expressing native or codon-optimized FVIII HC and VP1. For lettuce plants expressing 239

either native or codon-optimized CNTB-FVIII HC, chloroplast genomic DNA was digested with 240

HindIII and probed with DIG-labelled probe spanning the flanking region. All selected lines 241

showed the expected distinct hybridizing fragments and no untransformed fragment (Fig. 2A and 242

2B). The homoplasmic tobacco lines expressing native or codon optimized CNTB-VP1 243

sequences were already confirmed in previous study (Chan et al., 2016). Therefore, these data 244

confirm homoplasmy of all transplastomic lines; transgene expression levels should therefore be 245

attributed to translation efficiency and not transgene copy number. 246

Translation efficiency of native and codon-optimized genes in lettuce and tobacco 247

chloroplasts 248

Expression levels between native and codon-optimized genes in chloroplasts were 249

compared using immunoblot and densitometry assays. Early studies in this project compared 250

translation efficiency of the old algorithm (using only the most preferred codons) with the new 251

algorithm (using the psbA codon hierarchy) quantified by integrated density values (IDV) of 252

western blots (Fig. 2D). The CNTB-VP1 expression level in transplastomic plants using the old 253

algorithm for codon optimization was 2.7-3.1 fold lower than the native VP1 viral gene sequence 254

and the increase in VP1 expression was 77-111 fold higher using the new algorithm (Fig. 2D). 255

Therefore, the new algorithm of the codon optimizer program was used in all subsequent studies. 256

In order to correct for over or under exposure of western blots to X-ray film, data on variable 257

exposures were collected. In order to account for extreme variation in expression levels of native 258



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and codon optimized genes, serial dilutions of extracted proteins were loaded in each blot (Fig 259

3A and 3B, S4). In densitometry assay of lettuce expressing native and codon-optimized CNTB-260

FVIII HC which was also used for PRM, the concentration of FVIII HC from the codon-261

optimized gene (108.8 ~ 137.5 ug/g dry weight, DW) was 6.3 ~ 8.0 fold higher than that of the 262

native FVIII HC gene (16.9 ~ 17.4 µg/g DW) (Fig. S4). For tobacco plants expressing CNTB-263

VP1, the batch used for PRM mass spectrometry showed 91-125 fold difference between codon-264

optimized (11.3 µg/mg ~ 18.1 µg/mg) and native sequence (0.12 µg/mg ~ 0.15 µg/mg) (Fig. 3C 265

and S4). Based on these data, codon-optimized sequences obtained from our newly developed 266

codon optimizer program improved translation of transgenes to different levels, based on the 267

coding sequence. 268

To investigate the impact of codon optimization on transcript stability, northern blots 269

were performed using a probe for the psbA 5ʹ sequence (Fig. 4). Although loading controls show 270

equal amounts of total RNA in each lane based on ethidium bromide staining, higher or lower 271

level of the endogenous psbA transcript is observed among samples, suggesting subtle changes 272

in RNA loading. The mRNA levels of codon-optimized or native sequences for CNTB-FVIII 273

HC and CNTB-VP1 were normalized to endogenous psbA transcripts using densitometry and the 274

normalized ratios in each sample was compared. Northern blots indicated that the increase of 275

codon-optimized CNTB-FVIII HC and -VP1 accumulation is at the translational level rather than 276

RNA transcript accumulation. Several previous studies on expression of foreign genes have 277

shown a lack of variation or modest increases in transcript abundance but significant variation in 278

translation efficiency (Franklin et al., 2002; Gibsy et al 2011; Nakamura et al., 2016). Franklin et 279

al. (2002) reported a lack of variation in transcript abundance for GFP expression in 280

Chlamydomonas reinhardtii chloroplasts despite 80-fold increase in GFP protein accumulation 281



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of the codon optimized sequence. Even though there was a 3-fold increase in mRNA levels of 282

codon-optimized TGF-β3 when compared with the native sequence (Gibsy et al., 2011), the 283

greater part of the 75-fold increase in synthetic TGF-β sequence is attributed to enhanced 284

translation. A recent study also showed that compatibility of the 5ʹ-UTR and its coding sequence 285

increased efficient translation of codon-optimized sequences rather than mRNA abundance. 286

Absolute quantitation by PRM analysis 287

Expression levels of codon-optimized and native gene sequences were also quantified 288

using PRM mass spectrometry (Fig. 5). To select the optimal proteotypic peptides for PRM 289

analysis of the CNTB and FVIII HC sequences, we first performed a standard MS/MS analysis 290

(data not shown) of a tryptic digest of lettuce plants expressing CNTB-FVIII HC to choose 291

specific peptides. Expression of codon optimized FVIII HC was 5.4 or 5.8-fold higher than the 292

native sequence when the fold changes were normalized based on the house-keeping protein 293

peptides or SIS peptides (Fig. 5, 6A). Peptides chosen from CNTB showed minor variations in 294

fold changes based on the location of peptides and normalized with SIS or house-keeping protein 295

peptides from Rubsico (small or large subunits) or ATP synthase subunit beta (atpβ): 4.9 (or 4.5) 296

(IAYLTEAK), 5.2 (or 4.8) (IFSYTESLAGK), or 6.6 (or 6.1) (LCVWNNK). Peptides chosen 297

from FVIII HC also showed minor variations: 5.4 (or 5.0) (FDDDNSPSFIQIR), 5.7 (or 5.2) 298

(YYSSFVNMER) or 7.1 (or 6.6) (WTVTVEDGPTK) (Fig. 6A). The locations of these selected 299

peptides within CNTB-FVIII HC are shown in Supplemental Fig. S5. Please see raw data 300

included in the Supplemental Data Sets for more details. 301

Expression of codon optimized CTB-VP1 was 25.9 or 26.1 fold higher than the native 302

sequence when their fold changes were normalized based the SIS peptides or house-keeping 303

protein peptides (Fig. 5, 6B). Peptides chosen from CTB showed minimal variations in fold 304



14

changes based on their location: 22.5 (or 22.5) (LCVWNNK) to 26.1 (or 26.0) (IAYLTEAK) to 305

28.1 (or 28.0) (IFSYTESLAGK) (Fig. 6B). Linearity of the quantification range was also 306

investigated by spiking stable isotope-labeled standard (SIS) peptides in a constant amount of 307

plant digest (1:1:1:1 mix of all 4 types of plant materials) in a dynamic range covering 220 308

atomols to 170 fmol (values equivalent on column per injection). These results are reported in 309

detail in the Supplemental Data. For all six peptides, we observed an R2 value over 0.98. 310

Absolute quantitation can be achieved by spiking a known amount of the counterpart SIS 311

peptide into samples. For each counterpart, SIS peptide (34 fmol) was injected on column mixed 312

with protein digest (equivalent to protein extracted from 33.3 µg lyophilized leaf powder). By 313

calculating ratios of area under the curve (AUC) of SIS and endogenous peptides, we estimated 314

the endogenous peptide molarity, expressed as femtomole on column (Fig. 6). The mean of all 315

calculated ratios of femtomoles on column (6 and 3 peptides, CNTB-FVIII HC and CNTB-VP1, 316

respectively) for codon-optimized and native sequences is reported as the fold increase of protein 317

expression in codon-optimized constructs. The high reproducibility of the sample preparation 318

and PRM analysis is shown in Fig. 5. All peptide measurements were the result of four technical 319

replicates, two sample preparation replicates (from leaf powder to extraction to protein digestion) 320

and two mass spectrometry (MS) technical replicates. Coefficients of variation (%) among the 4 321

measurements per peptide ranged from 0.5% to 10% in all but two cases, where they were 16% 322

and 22%. 323

Ribosome profiling studies 324

Ribosome profiling uses deep sequencing to map “ribosome footprints”- mRNA 325

fragments that are protected by ribosomes from exogenous nuclease attack. The method provides 326

a genome-wide, high-resolution, and quantitative snapshot of mRNA segments occupied by 327



15

ribosomes in vivo (Ingolia et al., 2009). Total ribosome footprint abundance within an open 328

reading frame can provide an estimate of translational output, and positions at which ribosomes 329

slow or stall are marked by regions of particularly high ribosome occupancy. 330

To examine how codon optimization influenced ribosome behavior, we profiled 331

ribosomes from plants expressing the native and codon-optimized CNTB-FVIII HC and CNTB-332

VP1 transgenes. Fig. 7 shows the abundance of ribosome footprints as a function of position in 333

each transgene; footprint coverage on the endogenous chloroplast psbA and rbcL genes is shown 334

as a means to normalize the transgene data between the optimized and native constructs. 335

Ribosome footprint coverage was much higher in the codon-optimized VP1 sample than in the 336

native VP1 sample (Fig. 7A). However, the magnitude of this increase varies depending upon 337

how the data are normalized (Fig. 7C): the increase is 5-fold, 16-fold, or 1.5-fold when 338

normalized to total chloroplast ribosome footprints, psbA ribosome footprints, or rbcL ribosome 339

footprints, respectively. These numbers are considerably lower than the 22.5-28.1-fold increase 340

in VP1 protein abundance inferred from the quantitative mass spectrometry data. The topography 341

of ribosome profiles is generally highly reproducible among biological replicates (see for 342

example, rbcL and psbA in Fig. 7B) which are at the same developmental stage and grown under 343

the same conditions. In that context it is noteworthy that the peaks and valleys in the endogenous 344

psbA and rbcL genes are quite different in the native and optimized tobacco VP1 lines. It could 345

be envisaged that competition with the endogenous psbA 5ʹ-UTR could, in principle, reduce 346

translation of the endogenous psbA ORF. However, no such competition was observed for the 347

lettuce construct. In addition, the degree of competition would depend on the abundance of the 348

transgene mRNA. The abundance of the transgene mRNA was similar in the native and codon-349

optimized constructs, so competition via the psbA 5ʹ-UTR is unlikely to contribute to differences 350



16

in psbA ribosome occupancy in these lines. Many of the large peaks (presumed ribosome pauses) 351

observed in these endogenous genes, specifically in the native VP1 line, map to paired alanine 352

codons (asterisks in Fig. 7A). This suggests a limitation of alanine tRNA specifically in the 353

native VP1 line. Though the basis for this is unclear, it is conceivable that it has to do with minor 354

differences in age of the plants used for the analyses (2.5 months versus 2 months). It is also 355

conceivable that introduction of the transgene had an unanticipated effect on the expression of 356

the nearby gene encoding alanine tRNA. In the same vein, ribosome pause sites in the CNTB 357

region would be expected but the sites of the native and optimized VP1 constructs were not 358

similar. This global difference in ribosome behavior at alanine codons may well contribute to 359

differential transgene expression in the native and codon-optimized lines. 360

The total number of ribosome footprints in the FVIII gene decreased ~2-fold in the 361

codon-optimized line, whereas protein accumulation increased 4.5-6.6 fold. However, a major 362

ribosome pause can be observed near the 3ʹ end of the native transgene, followed by a region of 363

very low ribosome occupancy (see bracketed region in Fig. 7B). This ribosome pause maps to a 364

pair of CTC leucine codons, a codon that is almost not used in native psbA genes (see Fig. 1). 365

These results strongly suggest that the stalling of ribosomes at these leucine codons limits 366

translation of the downstream sequences and overall protein output, while also causing a buildup 367

of ribosomes on the upstream sequences. Thus, overall ribosome occupancy does not reflect 368

translational output in this case. Modification of those leucine codons in the codon-optimized 369

variant eliminated this ribosome stall and resulted in a much more even ribosome distribution 370

over the transgene (Fig. 7B, right). Taken together, the ribosome profiling data revealed dramatic 371

differences in ribosome dynamics between codon-optimized and native transgenes. Although 372

total ribosome occupancy did not reliably predict protein output from transgenes expressed in 373



17

chloroplasts, the detection of strong ribosome pauses at specific sites can provide insight into 374

rate-limiting steps that could be mitigated through sequence modifications. 375

Discussion 376

Past studies on transgene expression in chloroplasts reported abundant transcripts but 377

variable levels of translation based on the origin of coding sequence. Prokaryotic genes were 378

translated more efficiently than eukaryotic genes. Transcript abundance is attributed to high copy 379

number of transgenes and strength of the psbA promoter. Among >150 transgenes expressed in 380

chloroplasts, >75% utilized psbA regulatory sequences (Jin and Daniell, 2015; Daniell et al, 381

2016). In addition, three ribosome binding regions in the 5ˊ-UTR of psbA recruit ribosomes and 382

efficiently form translational initiation complex (Zou et al., 2003). Therefore, it is expected that 383

improvement of translation elongation of heterologous genes should increase transgene 384

expression. There is a drawback of using a codon table based on all chloroplast genes, which 385

assumes that all tRNA species are equally abundant. However such translational selection is not 386

possible (Surzycki et al., 2009). Therefore, in this study we developed a codon optimizer 387

program based on the codon usage of psbA genes across 133 plant species to increase expression 388

of heterologous genes in chloroplasts. 389

Codon optimization significantly enhances translation in chloroplasts 390

The psbA promoter and 5ˊ-UTR are most widely used regulatory sequences for transgene 391

expression in chloroplasts Among >115 transgenes expressed via the chloroplast genome, 84 use 392

the psbA regulatory sequence (Daniell et al., 2016 A, B; Jin and Daniell, 2015). A recent study 393

(Nakamura et al., 2016) shows the absence of any detectable translation when codons for the tat 394

coding sequence of HIV-1 were optimized using all 79 tobacco chloroplast mRNAs and 395

regulated by the psbA 5ʹ-UTR (Nakamura et al., 2016), but the same sequence was expressed 396



18

well using the phage T7 gene 10 5ʹ-UTR. However, when the 5ʹ psbA coding sequence was 397

inserted between the psbA 5ʹ-UTR and the tat sequence, translation was initiated. Therefore, 398

compatibility between the psbA regulatory element and codons is vital for initiation and 399

elongation during translation of heterologous genes (Nakamura et al., 2016). Therefore, when 400

heterologous genes are regulated by the psbA, codon optimization based on psbA codon usage 401

should facilitate the movement of ribosomes more efficiently from the translational initiation 402

complex than codon-optimized sequences based on any other chloroplast genes. 403

In this study, we developed and tested two new codon optimizer programs based on the 404

codon preference of psbA genes to improve the expression of heterologous genes in chloroplasts, 405

in concert with the psbA regulatory elements. The first “old” algorithm of the codon optimizer 406

was programmed to use only the most highly used codons, resulted in lower expression than the 407

native gene. The increase in expression of VP1 in chloroplasts between the old and new 408

algorithm is 77-111 fold. Therefore removal of rare codons and replacement with only highly 409

preferred codons did not help in enhancing translation when tRNA pools are limited. Therefore, 410

the new algorithm of the codon optimizer program was used in all subsequent studies. Therefore, 411

the “new” algorithm of the codon optimizer used the codon distribution hierarchy observed 412

among psbA genes. As a result, 105 rare codons out of 754 codons in the FVIII HC gene and 59 413

rare codons out of 302 codons in the VP1 gene were replaced with psbA preferentially used 414

codons. However, replaced codons are not identified as rare codons in codon tables using all 415

chloroplast genes. Therefore, total chloroplast codon table would have retained 75 rare codons 416

in FVIII-HC and 35 rare codons in the VP1 coding sequence. 417

Although we used a psbA-based codon optimization program to improve translation in 418

chloroplasts, many other factors, including the size and origin of heterologous genes and 419



19

compatibility of 5ʹ-UTR and its 5ʹ coding region were are important. The CNTB-fused native 420

sequence of human proinsulin (CNTB-Pins, ~22 kDa) was expressed up to 72% of total leaf 421

protein (Ruhlman et al., 2010) and the expression of ZZTEV-IGF-1 (Staphylococcus aureus Z 422

domains and TEV cleavage site fused to native human insulin-like growth factor 1 gene, ~26 423

kDa) was up to 32.4% of TSP (Daniell et al., 2009). However, human transforming growth 424

factor-β3 (TGF-β3, 13 kDa, 56% GC) was expressed up to 12% of leaf protein only after codon 425

optimization (Gisby et al., 2011). Also, expression of GFP (~26 kDa) increased ~80 fold after 426

codon optimization (Franklin et al., 2002). Therefore, proteins with shorter coding sequences are 427

not ideal to evaluate codon optimization concepts and other limitations in translation. 428

Consequently, a better understanding of codon usage and other rate-limiting steps (compatibility 429

with regulatory sequences, efficiency of translation initiation, elongation, and availability of 430

tRNAs) in translation is essential for successful expression of human or other eukaryotic coding 431

sequences. 432

Codon usage in psbA (our program) is different for preferred Arg, Asn, Gly, His, Leu and 433

Phe codons than those reported for 79 tobacco chloroplast mRNAs based on in vitro studies 434

(Nakamura and Sugiura, 2007). Preferred codons are decoded more rapidly than non-preferred 435

codons, presumably due to higher concentrations of corresponding tRNAs that recognize 436

preferred codons, which speeds up the elongation rate of protein synthesis (Yu et al., 2015). 437

Higher plant chloroplast genomes code for a conserved set of 30 tRNAs. This set is believed to 438

be sufficient to support translation machinery in chloroplasts (Lung et al., 2006). In the ribosome 439

profiling data for codon-optimized VP1, two major ribosome stalling sites correlated with an 440

unusually high concentration of serine codons (Fig. 7A). Five serine codons were clustered at 441

codons 71, 73, 75, 76 and 79, and three other serine codons were found at codons 178, 179 and 442



20

182. Two adjacent serines in each cluster, codons 75 and 76 (UCU-AGU), and codons 178 and 443

179 (UCC-UCU) (see triangles in Fig. 7A) show a high level of ribosome stalling. Thus, it may 444

be possible to further increase expression of the codon-optimized VP1 transgene by replacing 445

these codons with codons for a different but similar amino acid. 446

As seen in this study, the AT content of codon-optimized VP1 was marginally increased, 447

but the protein level of the optimized CNTB-VP1 increased significantly, up to 22.5-28.1 fold 448

(by PRM) and 91-125 fold (by western blot) over the native sequence when expressed in 449

chloroplasts. Therefore, several other factors play key roles in regulating efficiency of translation. 450

As observed in ribosome profiling studies of CNTB-VP1, the availability and density of specific 451

codons could severely impact translation. Similarly, FVIII HC ribosome footprint results showed 452

that ribosome pauses mapped to CTC leucine codons, which are almost not used in psbA genes. 453

This codon is also rarely used in the lettuce rbcL gene (2.44%) and is never used in tobacco rbcL. 454

Native FVIII HC uses the CTC codon as much as 15.28%, but the CTC codon was eliminated 455

from the codon-optimized sequence based on psbA codon usage. More detailed analysis of codon 456

frequency of the native FVIII HC and the psbA gene reveals further insight into rare codons; 457

GGG for Gly is used 2.3% in psbA but 11.63% in native HC; CTG for Leu is 3.7% in psbA but 458

26.39% in native HC; CCC for Pro is 1.9% vs 11.9%; CGG for Arg is 0.5% vs 10.81%; and 459

CTG for Val is 1.7% vs 25.49%. So, similar to the CTC codon, several other rare codons in 460

native human genes should have reduced translational efficiency in chloroplasts. In the process 461

of developing the codon optimizer, the cutoff value used for determination of codons was set at 5% 462

to eliminate rare codons. So, there is room to further modify the codon optimizer program. 463

New solution for quantitation of insoluble multimeric proteins 464



21

A major challenge is the lack of reliable methods to quantify insoluble proteins because 465

the only reliable method (ELISA) cannot be used due to aggregation or formation of multimeric 466

structures. CNTB fusion proteins expressed in chloroplasts form pentameric structures that are 467

highly resistant to detergents, and this hampers solubilization due to tight interactions between 468

CNTB monomers, mediated by 30 hydrogen bonds, 7 salt bridges and hydrophobic interactions 469

(Miyata et al., 2012). In our previous studies (Kwon et al., 2013; Shil et al., 2014; Kohli et al., 470

2014; Boyhan and Daniell, 2011), multimeric forms exist even after treatment with DTT, 471

detergents (SDS) and boiling. Also, acid (pH=2) could not completely dissociate CNTB 472

pentamers due to reformation of multimeric structures. Although such stability of pentamers is 473

ideal for oral drug delivery of CNTB fusion proteins, quantitation of dose continues to be a 474

major challenge. 475

Delivering accurate doses of protein drugs is a fundamental requirement for their clinical 476

use. Therefore, in this study we carried out parallel reaction monitoring (PRM) analysis for 477

absolute quantitation of CNTB-FVIII HC and CNTB-VP1 in plants carrying codon-optimized 478

and native sequences. Limitations in quantitation using western blots including protein 479

aggregation and inefficient transfer of large proteins to membranes, inadequate solubilization and 480

differential exposure to films were quite evident, resulting in unreliable quantification of drug 481

dosage in planta. Use of strong denaturing and reducing conditions in combination with optimal 482

enzymatic proteolysis conditions maximized solubilization of multimeric CNTB proteins. PRM 483

analysis has been broadly adopted in quantitative proteomics studies, e.g. biomarker discovery in 484

plasma, due to its high sensitivity, specificity and precise quantitation of specific protein targets 485

within complex protein matrices (Gallien et al., 2012). These qualities clearly show the 486

advantage of using PRM in the quantification of specific protein targets, independently of the 487



22

protein matrix source (e.g. plant extracts from tobacco or lettuce) or complexity. Moreover, the 488

development of a PRM assay for a handful of proteins can be achieved in a relatively short time 489

and at low cost (not considering the MS instrumentation). As a peptide-centric quantitation 490

methodology, it also offers robustness and versatility of protein extraction methods and keeping 491

the protein of interest in a native conformation is not required. However, it is intrinsically biased 492

by the enzymatic cleavage site access of the enzymes used for digestion. In order to overcome 493

this bias, we have used strong denaturing conditions (i.e. 2 % SDS) and buffers that favor 494

activity of the proteolytic enzymes (i.e. sodium deoxycholate-based buffers) (León et al., 2013). 495

For FVIII HC (Fig. 5 and 6), there was no significant variations in the values for fold increases 496

of codon-optimized over native sequences, which were determined by the peptides chosen for 497

quantification. In addition, the fold increases were very similar between two different 498

normalization approaches. Three peptides selected from the CNTB region (N-terminus of the 499

fusion protein) showed that the range of the fold increase was from 4.5 ~ 6.6 while the range was 500

5.0 ~ 7.1 for the peptides chosen from FVIII regions (C-terminus of the fusion protein). So 501

quantification results obtained from PRM analysis are consistent, irrespective of the selected 502

region of the fusion protein (N or C-terminus) or the component protein (CNTB or FVIII HC). 503

By using absolute quantified SIS peptides at identical concentrations in all samples and by 504

examining the entire length from N- to C-terminus, one could accurately quantify the absolute 505

amount of the target protein (Streng et al., 2016). Furthermore, the accuracy of PRM assays in 506

this study was further consolidated by using two different normalization methods including SIS 507

peptides and peptides for house-keeping proteins (large or small subunit proteins of Rubisco and 508

ATP synthase β subunit). Incomplete/cleaved proteins can be detected by using targeted peptide 509

located closer to the C, N terminus or in the mid-regions. Quantification results obtained from 510



23

PRM analysis of both CNTB fusion proteins in our study are consistent, irrespective of the 511

selected region of the fusion protein (N or C-terminus or elsewhere), offers data for reliable 512

quantitation. Also, the same three CNTB peptides for CNTB-VP1 showed consistent fold 513

increases, ranging from 22.5 ~ 28.1. PRM analysis is better than western blots because it 514

eliminates variation introduced by mobility and transfer of different size proteins and saturation 515

of antibody probes. Overall, the PRM workflow included selection of the proteotypic peptides 516

from CNTB and FVIII HC sequences; and synthesis of the counterpart SIS peptides 517

(Supplemental Fig. S6). Six peptides were selected and scheduled for PRM analysis on the 518

Qexactive mass spectrometer, based on observed retention time (RT) on the chromatography 519

with a window of ± 5 min and mass over charge (m/z) of double and/or triple charge state of 520

these peptides. This double way of targeting the selection of precursor ions, in addition to the 521

high resolution of the Qexactive MS, contributes to the high specificity of the assay. The PRM 522

data analysis, post-acquisition, also offers a high specificity to the assay. The five most intense 523

fragment ions, with no clear contaminant contribution from the matrix, are then selected for the 524

quantification of the peptide. The confidence of the fragment ion assignment by the 525

bioinformatics tool used, i.e. Skyline (MacLean et al., 2010) is finally achieved by the 526

comparison of the reference MS/MS spectra and the RT profiles, generated with each of the 527

counterpart SIS peptides. The high sensitivity, specificity, versatility and robustness of PRM 528

offer a new opportunity for characterizing translational systems in plants. 529

Conclusions 530

This study explored heterologous gene expression utilizing chloroplast genome 531

sequences, ribosome profiling and targeted mass spectrometry (MS) to enhance our 532

understanding of the synthesis of valuable biopharmaceuticals in chloroplasts. Targeted 533



24

proteomic quantification by mass spectrometry showed that codon optimization increases 534

translation efficiency 4.5-28.1 fold based on the coding sequence, validating this approach for 535

the first time for quantitation of protein drug dosage in plant cells. The lack of reliable methods 536

to quantify insoluble proteins due to aggregation or formation of multimeric structures is a major 537

challenge. Both biopharmaceuticals used in this study are CNTB fusion proteins that form 538

pentamers, which is a requirement for their binding to intestinal epithelial GM1 receptors. Such a 539

multimeric structure excludes the commonly used ELISA for quantitation of dosage. However, 540

delivering accurate doses of protein drugs is a fundamental requirement for their clinical use and 541

this important goal was accomplished in this study. Indeed, plant biomass generated in this study 542

has resulted in the development of a polio booster vaccine that has been validated by the Centers 543

for Disease Control and Prevention, a timely invention to meet the World Health Organization 544

requirement to withdraw the current oral polio vaccine, which causes severe polio in outbreak 545

areas, in April 2016 (Chan et al 2016). 546

Such increase of codon-optimized protein accumulation is at the translational level rather 547

than any impact on transcript abundance. The codon optimizer program increases transgene 548

expression in chloroplasts in both tobacco and lettuce, with no species specificity. In contrast to 549

previous in vitro studies, first in depth in vivo studies of heterologous gene expression using a 550

wealth of newly sequenced chloroplast genomes helped us to understand the codon optimization 551

process. While removal of rare codons is very important, replacing those with the most highly 552

used psbA codons indeed decreased translation efficiency. So, the key factor in enhancing 553

translation is replacement of rare codons following the hierarchy of a highly expressed gene. 554

Ribosome footprints obtained using profiling studies did not increase proportionately with VP1 555

translation or even decreased after FVIII codon optimization, but it is a valuable tool for 556



25

diagnosing rate-limiting steps in translation. A major ribosome pause at CTC leucine codons, a 557

rarely used codon in chloroplasts, was eliminated from the native gene after codon optimization. 558

Ribosome stalls observed at clusters of other codons in codon-optimized genes provide 559

opportunities for further optimization. These observations provide further insight into limitations 560

in chloroplast translation and approaches to address this in future studies. 561

Materials and methods 562

Codon optimization 563

To maximize the expression of heterologous genes in chloroplasts, a chloroplast codon optimizer 564

program was developed based on the codon preference of psbA genes across 133 seed plant 565

species. All sequences were downloaded from the National Center for Biotechnology 566

Information (NCBI, http://www.ncbi.nlm.nih.gov/genomes/ GenomesGroup. 567

cgi?taxid=2759&opt=plastid). The usage preference among synonymous codons for each amino 568

acid was determined by analyzing a total of 46,500 codons from 133 psbA genes. The 569

optimization algorithm (Chloroplast Optimizer v2.1) was made to facilitate changes from rare 570

codons to codons that are frequently used in chloroplasts using JAVA. 571

Creation of transplastomic lines 572

The native sequence of the FVIII heavy chain (HC) was amplified using the pAAV-TTR-573

hF8-mini plasmid (Sherman et al., 2014) as the PCR template. The codon-optimized HC 574

sequence obtained using Codon Optimizer v2.1 was synthesized by GenScript (Piscataway, NJ, 575

USA). The native VP1 gene (906 bp) of Sabin 1 (provided by Dr. Konstantin Chumakov, FDA) 576

was used as the template for PCR amplification. The codon-optimized VP1 sequence was also 577

synthesized by GenScript. Amplified and synthetic gene sequences were cloned into chloroplast 578

transformation vectors pLSLF and pLD-utr for Lactuca sativa and Nicotiana tabacum cv. Petite 579



26

Havana, respectively. Sequence-confirmed plasmids were used for bombardment to create 580

transplastomic plants as described previously (Verma et al., 2008). Transplastomic lines were 581

confirmed using Southern blot analysis as described previously (Verma et al., 2008) except for 582

probe labeling and detection, for which DIG high prime DNA labeling and detection starter kit II 583

(Roche, cat no. 11585624910) was used. 584

Evaluation of translation 585

To compare the level of protein expression between native and codon-optimized sequences, 586

immunoblots and densitometric assays were performed using anti-CNTB antibody. For total 587

plant protein, powdered lyophilized plant cells were suspended in extraction buffer (100 mM 588

NaCl, 10 mM EDTA, 200 mM Tris-Cl pH 8.0, 0.05% (v/v) Tween-20, 0.1% SDS, 14 mM β-ME, 589

400 mM sucrose, 2 mM PMSF, and proteinase inhibitor cocktail) in a ratio of 10 mg per 500 µL 590

and incubated on ice for 1 h for rehydration. Suspended cells were sonicated (pulse on for 5 s 591

and pulse off for 10 s, sonicator 3000, Misonix) after vortexing (~30 s). After Bradford assay, 592

equal amounts of homogenized protein were loaded and separated on SDS-polyacrylamide with 593

known amounts of CNTB protein standard. To detect CNTB fusion proteins, anti-CNTB 594

polyclonal antibody (GenWay Biotech Inc., San Diego, CA) was diluted 1:10,000 in 1X PBST 595

(0.1 % Tween-20) and then membranes were probed with goat anti-rabbit IgG-HRP secondary 596

antibody (Southern Biotechnology, 4030-05) diluted 1:4,000 in 1X PBST. For loading controls, 597

protein-blotted membrane was stained with Ponceau S (Sigma, P-3504) prior to immunoprobing 598

with anti-CNTB antibody and anti-RbcL antibody (Agrisera, AS03 037, 1 in 5,000) was used on 599

the same blots after stripping anti-CNTB antibody. Chemiluminescent signals were developed on 600

X-ray films, which were used for quantitative analysis with Image J software (IJ 1.46r; NIH). 601

Evaluation of transcripts 602



27

Total RNA was extracted from leaves of plants grown in agar medium in tissue culture 603

room using an easy-BLUETM total RNA extraction kit (iNtRON, cat no. 17061). For the RNA 604

gel blot, equal amounts of total RNA were separated on a 0.8% agarose gel (containing 1.85% 605

formaldehyde and 1X MOPS) and blotted onto a nylon membrane (Nytran SPC; Whatman, 606

Buckinghamshire, UK). For northern blot, the PCR-amplified product from psbA 5ʹ-UTR region 607

of chloroplast transformation plasmid was used as the probe. Hybridization signals on 608

membranes were detected using a DIG labeling and detection kit as described above. 609

Lyophilization 610

Confirmed homoplasmic lines were transferred to a temperature- and light-controlled 611

greenhouse. Mature leaves from fully grown transplastomic plants were harvested and stored at -612

80°C before lyophilization. To freeze-dry plant leaf materials, frozen, crumbled small leaf pieces 613

were sublimated under 400 mTorr vacuum while increasing the chamber temperature from -40°C 614

to 25°C for 3 days (Genesis 35XL, VirTis SP Scientific). Dehydrated leaves were powdered 615

using a coffee grinder (Hamilton Beach) at maximum speed; tobacco was ground 3 times for 10 616

sec each and lettuce was ground 3 times for 5 sec. Powdered leaves were stored in containers 617

under air-tight and moisture-free conditions at room temperature with silica gel. 618

Protein extraction and sample preparation for mass spectrometry analysis 619

Total protein was extracted from 10 mg of lyophilized leaf powder by adding 1 mL 620

extraction buffer (2% SDS, 100 mM DTT, 20 mM TEAB). Lyophilized leaf powder was 621

incubated for 30 min at RT with sporadic vortexing to allow rehydration of plant cells. 622

Homogenates were then incubated for 1 h at 70 ºC, followed by overnight incubation at RT 623

under constant rotation. Cell wall/membrane debris was pelleted by centrifugation at 14,000 rpm 624

(approx. 20,800 rcf). The procedure was performed in duplicate. 625



28

All protein extracts (100 µL) were enzymatically digested with 10 µg trypsin/Lys-C 626

(Promega) on a centrifugal device with a filter cut-off of 10 kDa (Vivacon) in the presence of 0.5% 627

sodium deoxycholate, as previously described (León et al., 2013). After digestion, sodium 628

deoxycholate was removed by acid precipitation with 1% (final concentration) trifluoroacetic 629

acid. Stable isotope-labeled standard (SIS) peptides (>97% purity, C-term Lys and Arg as Lys U-630

13C6;U-15N2 and Arg U-13C6;U-15N4, JPT Peptide Technologies) were spiked into the 631

samples prior to desalting. Samples were desalted prior to MS analysis with OligoR3 stage-tips 632

(Applied Biosystems). The initial protein extract (10 µL) was desalted on an OligoR3 stage tip 633

column. Desalted material was then dried on a speed vacuum device and suspended in 6 µL of 634

0.1% formic acid in water. MS analysis was performed in duplicate by injecting 2 µL of desalted 635

material into the column. 636

PRM mass spectrometry analysis and data analysis 637

Liquid chromatography-coupled targeted mass spectrometry analysis was performed by 638

injecting the column with 2 µL of peptide, corresponding to the amount of total protein extracted 639

and digested from 33.3 µg of lyophilized leaf powder, with 34 fmol of each SIS peptide spiked in. 640

Peptides were separated using an Easy-nLC 1000 (Thermo Scientific) on a home-made 30 cm x 641

75 µm i.d. C18 column (1.9 µm particle size, ReproSil, Dr. Maisch HPLC GmbH). Mobile 642

phases consisted of an aqueous solution of 0.1% formic acid (A) and 90% acetonitrile and 0.1% 643

formic acid (B), both HPLC grade (Fluka). Peptides were loaded on the column at 250 nL/min 644

with an aqueous solution of 4% solvent B. Peptides were eluted by applying a non-linear 645

gradient for 4-7-27-36-65-80%B in 2-50-10-10-5 min, respectively. 646

MS analysis was performed using the parallel reaction monitoring (PRM) mode on a 647

Qexactive mass spectrometer (Thermo Scientific) equipped with a nanospray FlexTM ion source 648



29

(Gallien et al., 2012). Isolation of targets from the inclusion list with a 2 m/z window, a 649

resolution of 35,000 (at m/z 200), a target AGC value of 1 x 106, and a maximum filling time of 650

120 ms. Normalized collision energy was set at 29. Retention time schedules were determined by 651

the analysis of SIS peptides under equal nanoLC chromatography. A list of target precursor ions 652

and retention time schedule is reported in the Supplementary Information. PRM data analysis 653

was performed using Skyline software (MacLean et al., 2010). 654

Ribosome profiling 655

Second and third leaves from the top of the plant were harvested for ribosome profiling. 656

Lettuce plants were approximately 2 months old. Tobacco plants were 2.5 or 2 months old, for 657

native and codon-optimized VP1 constructs, respectively. Leaves were harvested at noon and 658

flash frozen in liquid nitrogen. Ribosome footprints were prepared as described in Zoschke et al 659

(2013) except that ribonuclease I was substituted for micrococcal nuclease. Ribosome footprints 660

were converted to a sequencing library with the NEXTflex Illumina Small RNA Sequencing Kit 661

v2 (BIOO Scientific, 5132-03). rRNA contaminants were depleted by subtractive hybridization 662

after first strand cDNA synthesis using biotinylated oligonucleotides corresponding to abundant 663

rRNA contaminants observed in pilot experiments. Samples were sequenced at the University of 664

Oregon Genomics Core Facility. Sequence reads were processed with cutadapt to remove 665

adapter sequences and bowtie2 with default parameters to align reads to the engineered 666

chloroplast genome sequence. 667

Acknowledgements 668

Authors thank Mark Yarmarkovich for help with developing the codon optimization algorithms, 669

Nick Stiffler for help with the bioinformatic analysis of ribosome profiling data, and Non 670

Chotewutmontri for helpful discussions. This work was supported by NIH grants R01 HL107904, 671



30

R01 HL109442, R01 EY 024564 and Bill and Melinda Gates Foundation grant OPP1031406 to 672

Henry Daniell and NSF grant IOS-1339130 to Alice Barkan. 673

Conflict of interest statement 674

Henry Daniell, has several patents in the field of chloroplast genetic engineering and production 675

of biopharmaceuticals in chloroplasts. Google Scholar link is provided 676

http://scholar.google.com/citations?user=7sow4jwAAAAJ&hl=en for full disclosure of these 677

patents. However, he has no specific financial conflict of interest to declare. 678

679

Figure legends 680

Figure 1: Development of a codon optimization algorithm for expression of heterologous 681

genes in plant chloroplasts. A, Process to develop the codon optimization algorithm. Sequence 682

data of psbA genes from 133 plant species collected from NCBI and their codon preferences 683

were analyzed. Finally, the codon optimizer was developed using Java. B, Codon preference 684

table. Codon preference is indicated by the percentage of use for each amino acid. Black and 685

underlined codons indicate codons that were not used when optimizing sequences due to their 686

low usage frequency among synonymous codons (less than 5% use or, for amino acids with 6 687

synonymous codons – leucine, serine and arginine – the two codons used least frequently). 688

Figure 2: Construction of chloroplast vectors using native or codon-optimized genes, 689

evaluation of homoplasmy and transgene expression. A, Lettuce or tobacco chloroplast vector 690

maps. Prrn, rRNA operon promoter; aadA, aminoglycoside 3´-adenylytransferase gene; PpsbA, 691

promoter and 5´-UTR of psbA gene; CNTB, coding sequence of cholera non-toxic B subunit; 692

FVIII HC, factor 8 heavy chain native (N) or codon optimized (CN) using the new algorithm; 693

TpsbA, 3´-UTR of the psbA gene; trnI, isoleucyl-tRNA; trnA, alanyl-tRNA. B and C, Southern 694



31

blot analysis of homoplasmic lines. Total genomic DNA (3 µg) from untransformed (UT), native 695

(N) or codon-optimized CNTB-FVIII HC (new algorithm - CN) was digested with HindIII and 696

separated on a 0.8% agarose gel, blotted onto a Nytran membrane and probed with BamHI 697

fragment (SB-P). Lanes 1-4; four independent transplastomic lines. D, Comparison of expression 698

level of CNTB-VP1 between transplastomic lines expressing the native (N) or codon-optimized 699

genes using the old (CO) or new (CN) algorithm. Total extracted proteins were loaded as 700

indicated protein concentrations and were probed with anti-CNTB antibody. L.s., Lactuca sativa; 701

N.t., Nicotiana tabacum; CNTB, standard protein of cholera non-toxic B subunit; IDV, 702

integrated density values. 703

Figure 3: Quantitation of native or codon-optimized CNTB-FVIII HC or CNTB-VP1 gene 704

expression using western blots. Extracted leaf proteins were resolved on gradient (4%-20%) 705

SDS-PAGE and probed with anti-CNTB antibody (1 in 10,000). For loading control, the same 706

membranes were stripped and re-probed with anti-RbcL antibody (1 in 5,000). A, Lettuce leaf 707

protein extracts (5 or 10 ug) expressing CNTB-FVIII HC or untransformed. For loading controls 708

Ponceau S staining of membrane prior to western blot or re-probed blot with the large subunit of 709

Rubisco (RbcL) is provided. B, Serial dilution of the native (5-20ug) or codon optimized (1-4 ug) 710

CNTB-FVIII HC lettuce leaf extracts. C, Serial dilution of the native (2-8ug) or codon optimized 711

(0.1-0.4 ug) CNTB-VP1 tobacco leaf extracts. UT, untransformed wild type; N, native sequence; 712

codon-optimized with old algorithm (CO) or new algorithm (CN). CNTB standard proteins (1-6 713

ng). L.s., Lactuca sativa; N.t., Nicotiana tabacum. 714

Figure 4: Northern analysis of transplastomic lines: Transgene transcripts of CNTB-FVIII 715

HC (A) or CNTB-VP1 (B) were probed with 200 bp of lettuce psbA 5ʹ UTR (for FVIII HC) or 716

tobacco psbA 5ʹ UTR (for VP1) regulatory sequences, respectively. Lower and upper transcripts 717



32

represent the endogenous psbA gene and CNTB-FVIII or CNTB-VP1 transgenes, respectively. 718

Ethidium bromide (EtBr) stained gels are included for evaluation of equal loading. UT, 719

untransformed wild type; N, native sequence; CN, codon-optimized sequence using the new 720

algorithm. 721

Figure 5: PRM mass spectrometry analysis of CNTB-FVIII and CNTB-VP1 proteins at N- 722

to C-terminal protein sequences. Y axis shows molarity (fmol on column) of peptides from 723

CTB-FVIII HC or CTB-VP1 in codon-optimized or native genes. CNTB: peptide 1, 724

IFSYTESLAGK; peptide 2, IAYLTEAK; peptide 3, LCVWNNK. FVIII: peptide 4, 725

FDDDNSPSFIQIR; peptide 5, WTVTVEDGPTK; peptide 6, YYSSFVNMER. The median of 4 726

technical replicates is presented for each sample. Circle (●), native sequences; square (■), codon-727

optimized sequences using the new algorithm. 728

Figure 6: Fold change (increase) of CNTB-FVIII HC or CNTB-VP1 proteins based on 729

targeted MS analysis of CNTB and HC peptides. The reported data represent the median of 730

the results from six and three peptides from CNTB-FIII HC (A) and CNTB-VP1 (B), 731

respectively. Y axis represents the fold change increase (based on measured fmol on column) of 732

peptides from plant materials expressing genes codon-optimized using the new algorithm (CO) 733

with respect to plant materials expressing native sequence (CN). CNTB: peptide 1, 734

IFSYTESLAGK; peptide 2, IAYLTEAK; peptide 3, LCVWNNK. FVIII HC: peptide 4, 735

FDDDNSPSFIQIR; peptide 5, WTVTVEDGPTK; peptide 6, YYSSFVNMER. Stable isotope 736

labeled standard (SIS) normalized values represent fold change as a ratio to each spiked SIS 737

peptide. Housekeeping (HK) protein normalization values represent fold change as a normalized 738

ratio to Rubisco large or small subunit, ATP synthase CF1 beta subunit protein peptides. For 739

more details, see supplementary data on peptide ratio results for CNTB-FVIII and CNTB-VP1. 740



33

Figure 7: Ribosome profiling data from transplastomic plants expressing native and codon-741

optimized VP1 or FVIII HC. Read coverage for native transgenes (N), codon-optimized 742

transgenes with new algorithm (CN) and the endogenous psbA and rbcL genes are displayed with 743

the Integrated Genome Viewer (IGV). A, Data from tobacco leaves expressing native and codon-744

optimized VP1 transgenes. Asterisks mark each pair of consecutive alanine codons in the data 745

from the native line. The + symbol marks three consecutive alanine codons. Many strong 746

ribosome pause sites in the plants expressing native VP1 map to paired alanine codons, whereas 747

this is not observed in the codon-optimized line. Triangles mark each pair of consecutive serine 748

codons in the codon-optimized line. A major ribosome stall maps to a region harboring five 749

closely spaced serine codons in the codon-optimized VP1 gene. B, Data from lettuce plants 750

expressing the native and codon-optimized FVIII HC transgenes. A major ribosome stall in the 751

native FVIII HC gene maps to a pair of adjacent CTC leucine codons, a codon that is not used in 752

the native psbA gene. Ribosome footprint coverage is much more uniform on the codon-753

optimized transgene. C, Absolute and relative ribosome footprints counts. 754

755

Supplemental Data 756

Supplemental Data - Supplemental Dataset (codon usage table and MS spectrometry data) 757

Supplemental Figure S1. Sequences of native and codon-optimized FVIII HC or VP1 genes. 758

Supplemental Figure S2. Comparison of native and codon-optimized (new and old) sequences. 759

Supplemental Figure S3. Three different codon tables for the expression 760 of heterologous genes in chloroplasts. 761

Supplemental Figure S4. Plot of integrated density values (IDVs) for quantification of CNTB-FVIII HC 762

(A) and CNTB-VP1 (B) based on standard curves. 763

Supplemental Figure S5. Peptide sequences used for targeted MS. 764



34

Supplemental Figure S6. Comparison of CNTB-FVIII HC and VP1 by PRM analysis. 765

766

767

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A

B

Collection of sequence data from psbA genes from 133 plant species

Analysis of codon usage

Development of algorithm and software

Figure 1. Development of a codon optimization algorithm for expression of

heterologous genes in plant chloroplasts. A, Process to develop the codon

optimization algorithm. Sequence data of psbA genes from 133 plant species collected

from NCBI and their codon preferences were analyzed. Finally, the codon optimizer

was developed using Java. B, Codon preference table. Codon preference is indicated by

the percentage of use for each amino acid. Black and underlined codons indicate

codons that were not used when optimizing sequences due to their low usage frequency

among synonymous codons (less than 5% use or, for amino acids with 6 synonymous

codons – leucine, serine and arginine – the two codons used least frequently).

TTT (71.9%) F TCT (43.3%) S TAT (52.6%) Y TGT (85.8%) C

TTC (28.1%) F TCC (12.8%) S TAC (47.4%) Y TGC (14.2%) C

TTA (26.2%) L TCA (5.6%) S TAA (100%) STOP TGA (0%) STOP

TTG (22.5%) L TCG (2%) S TAG (0%) STOP TGG (100%) W

CTT (20.5%) L CCT (65.8%) P CAT (47.9%) H CGT (54.1%) R

CTC (0.1%) L CCC (1.9%) P CAC (52.2%) H CGC (17.8%) R

CTA (27.1%) L CCA (27.8%) P CAA (80.4%) Q CGA (8.4%) R

CTG (3.7%) L CCG (4.5%) P CAG (19.6%) Q CGG (0.5%) R

ATT (57.5%) I ACT (58.7%) T AAT (47.4%) N AGT (22.0%) S

ATC (34.0%) I ACC (30.9%) T AAC (52.6%) N AGC (14.7%) S

ATA (8.6%) I ACA (9.6%) T AAA (84.4%) K AGA (12.3%) R

ATG (100%) M ACG (0.8%) T AAG (15.6%) K AGG (6.9%) R

GTT (44.8%) V GCT (68.7%) A GAT (81.0%) D GGT (67.2%) G

GTC (2.2%) V GCC (7.0%) A GAC (19.0%) D GGC (13.0%) G

GTA (51.3%) V GCA (19.4%) A GAA (75.0%) E GGA (17.6%) G

GTG (2%) V GCG (4.9%) A GAG (25.0%) E GGG (2.3%) G



FVIII HC (CN)

HindIII HindIII

trnA trnI

BamHI BamHI SB-P

TpsbA trnA Prrn aadA PpsbA trnI

HindIII HindIII

HindIII

TpsbA trnA Prrn aadA PpsbA trnI

Untransformed chloroplast genome

Chloroplast transformation vector

Transformed chloroplast genome

A

B C

FVIII HC (N)

VP1 (N)

VP1 (CN)

HindIII HindIII

HindIII AflIII

AflIII AflIII

VP1 (N)

VP1 (CN)

CNTB FVIII HC (N)

CNTB FVIII HC (CN)

AflIII AflIII

AflIII AflIII

CNTB CNTB

CNTB CNTB

CNTB

CNTB

Figure 2. Construction of chloroplast vectors using native or codon-optimized genes, evaluation of homoplasmy and transgene expression. A, Lettuce or tobacco

chloroplast vector maps. Prrn, rRNA operon promoter; aadA, aminoglycoside 3´-adenylytransferase gene; PpsbA, promoter and 5´-UTR of psbA gene; CNTB, coding sequence of

cholera non-toxic B subunit; FVIII HC, factor 8 heavy chain native (N) or codon optimized (CN) using the new algorithm; TpsbA, 3´-UTR of the psbA gene; trnI, isoleucyl-tRNA;

trnA, alanyl-tRNA. B and C, Southern blot analysis of homoplasmic lines. Total genomic DNA (3 µg) from untransformed (UT), native (N) or codon-optimized CNTB-FVIII HC

(new algorithm - CN) was digested with HindIII and separated on a 0.8% agarose gel, blotted onto a Nytran membrane and probed with BamHI fragment (SB-P). Lanes 1-4; four

independent transplastomic lines. D, Comparison of expression level of CNTB-VP1 between transplastomic lines expressing the native (N) or codon-optimized genes using the old

(CO) or new (CN) algorithm. Total extracted proteins were loaded as indicated protein concentrations and were probed with anti-CNTB antibody. L.s., Lactuca sativa; N.t.,

Nicotiana tabacum; CNTB, standard protein of cholera non-toxic B subunit; IDV, integrated density values.

1 2 3 4 UT

9.1 kb

10.3 kb

3.3 kb

CNTB-FVIII HC (CN, L.s.)

9.1 kb

10.1 kb

2.2 kb

1 2 3 4 UT

CNTB-FVIII HC (N, L.s.) D

CNTB (ng) (ug)

N 2

37

50

75

15

20 25

20 0.4 10 30 0.6 15 CO CN N

4 6 kDa

1.02 0.75 1.10 0.71 1.59 1.15 (x104) (IDV)

CNTB-VP1 (N.t.)

CO CN



50

50 75

100 150 250

25

37

20 15 10

CNTB-FVIII HC (L.s.)

N CN UT N CN UT 5 ug 10 ug

1 2 4

CNTB(ng)

50 RbcL

CNTB-FVIII HC

50

75 100 150 250

25

37

20 15 10

50

50

75 100 150 250

25

37

20 15 10

50

10 20 5

CNTB-FVIII HC (L.s.)

2 4 1 (µg) UT 20

N CN

4 8 2

CNTB-VP1 (N.t.)

0.2 0.4 0.1 (µg) UT 8

N CN

1 2 4

CNTB(ng)

2 4 6 CNTB(ng)

CNTB-VP1

CNTB-FVIII HC

RbcL

RbcL

C

A B

Figure 3. Quantitation of native or codon-optimized CNTB-FVIII HC or

CNTB-VP1 gene expression using western blots. Extracted leaf proteins were

resolved on gradient (4%-20%) SDS-PAGE and probed with anti-CNTB antibody

(1 in 10,000). For loading control, the same membranes were stripped and re-

probed with anti-RbcL antibody (1 in 5,000). A) Lettuce leaf protein extracts (5 or

10 ug) expressing CNTB-FVIII HC or untransformed. For loading controls

Ponceau S staining of membrane prior to western blot or re-probed blot with the

large subunit of Rubisco (RbcL) is provided. B) Serial dilution of the native (5-

20ug) or codon optimized (1-4 ug) CNTB-FVIII HC lettuce leaf extracts. C)

Serial dilution of the native (2-8ug) or codon optimized (0.1-0.4 ug) CNTB-VP1

tobacco leaf extracts. UT, untransformed wild type; N, native sequence; codon-

optimized with old algorithm (CO) or new algorithm (CN). CNTB standard proteins

(1-6 ng). L.s., Lactuca sativa; N.t., Nicotiana tabacum.

Anti-CNTB

Anti-RbcL

Ponceau S

Anti-CNTB

Anti-RbcL



N CN UT

2.5 ug B

CN UT

rRNA stained with EtBr

Endogenous psbA

CNTB-FVIII HC

rRNA stained with EtBr

A

N

4 ug

Endogenous psbA

CNTB-VP1

Figure 4. Northern analysis of transplastomic lines: Transgene transcripts of CNTB-FVIII HC (A) or CNTB-VP1 (B)

were probed with 200 bp of lettuce psbA 5ʹ UTR (for FVIII HC) or tobacco psbA 5ʹ UTR (for VP1) regulatory sequences,

respectively. Lower and upper transcripts represent the endogenous psbA gene and CNTB-FVIII or CNTB-VP1 transgenes,

respectively. Ethidium bromide (EtBr) stained gels are included for evaluation of equal loading. UT, untransformed wild

type; N, native sequence; CN, codon-optimized sequence using the new algorithm.



CNTB-FVIII HC protein

CNTB-VP1 protein

CV%:1.7

CV%:5.63

CV%:1.9

CV%:2.9

CV%:22

CV%:0.6

CV%:7.5

CV%:1.2

CV%:8.3

CV%:2.8 CV%:5.3

CV%:17

CV%:6.7

CV%:2.4

CV%:1.9

CV%:1.4

CV%:2.1

CV%:10

Figure 5. PRM mass spectrometry analysis of CNTB-FVIII and CNTB-VP1 proteins at N- to C-terminal protein sequences. Y

axis shows molarity (fmol on column) of peptides from CTB-FVIII HC or CTB-VP1 in codon-optimized or native genes. CNTB:

peptide 1, IFSYTESLAGK; peptide 2, IAYLTEAK; peptide 3, LCVWNNK. FVIII: peptide 4, FDDDNSPSFIQIR; peptide 5,

WTVTVEDGPTK; peptide 6, YYSSFVNMER. The median of 4 technical replicates is presented for each sample. Circle (●), native

sequences; square (■), codon-optimized sequences using the new algorithm.



Figure 6. Fold change (increase) of CNTB-FVIII HC or CNTB-VP1 proteins based on targeted MS analysis of CNTB and HC

peptides. The reported data represent the median of the results from six and three peptides from CNTB-FIII HC (A) and CNTB-VP1

(B), respectively. Y axis represents the fold change increase (based on measured fmol on column) of peptides from plant materials

expressing genes codon-optimized using the new algorithm (CO) with respect to plant materials expressing native sequence (CN).

CNTB: peptide 1, IFSYTESLAGK; peptide 2, IAYLTEAK; peptide 3, LCVWNNK. FVIII HC: peptide 4, FDDDNSPSFIQIR; peptide

5, WTVTVEDGPTK; peptide 6, YYSSFVNMER. Stable isotope labeled standard (SIS) normalized values represent fold change as a

ratio to each spiked SIS peptide. House keeping (HK) protein normalization values represent fold change as a normalized ratio to

Rubisco large subunit (TFQGPPHGIQV and WSPELAAACEV) or small subunit (YETLSYLPPLSDEALSK), ATP synthase CF1 beta

subunit (FVQAGSEVSALLGR) protein peptides. For more details see supplementary data on peptide ratio results for CNTB-FVIII

and CNTB-VP1.

A B

Mean 5.8

Mean 25.9 Mean 26.1

Mean 5.4

Rat

io C

N/

N (

fmo

l on

co

lum

n)

Rat

io C

N/

N (

fmo

l on

co

lum

n)



A

B

C

Figure 7. Ribosome profiling data from

transplastomic plants expressing native and

codon-optimized VP1 or FVIII HC. Read

coverage for native transgenes (N), codon-

optimized transgenes with new algorithm (CN)

and the endogenous psbA and rbcL genes are

displayed with the Integrated Genome Viewer

(IGV). A, Data from tobacco leaves expressing

native and codon-optimized VP1 transgenes.

Asterisks mark each pair of consecutive alanine

codons in the data from the native line. The +

symbol marks three consecutive alanine codons.

Many strong ribosome pause sites in the plants

expressing native VP1 map to paired alanine

codons, whereas this is not observed in the

codon-optimized line. Triangles mark each pair

of consecutive serine codons in the codon-

optimized line. A major ribosome stall maps to a

region harboring five closely spaced serine

codons in the codon-optimized VP1 gene. B,

Data from lettuce plants expressing the native

and codon-optimized FVIII HC transgenes. A

major ribosome stall in the native FVIII HC gene

maps to a pair of adjacent CTC leucine codons, a

codon that is not used in the native psbA gene.

Ribosome footprint coverage is much more

uniform on the codon-optimized transgene. C,

Absolute and relative ribosome footprints

counts.



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Boyhan D, Daniell H (2011) Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery offunctional insulin and C-peptide. Plant Biotechnol J 9:585-598


Birch-Machin I, Newell CA, Hibberd JM, Gray JC (2004) Accumulation of rotavirus VP6 protein in chloroplasts of transplastomictobacco is limited by protein stability. Plant Biotechnol J 2:261-270


Chan HT, Daniell H (2015) Plant-made oral vaccines against human infectious diseases-Are we there yet? Plant Biotechnol J13:1056-1070


Chan HT, Xiao Y, Weldon WC, Oberste SM, Chumakov K, Daniell H (2016) Cold chain and virus free chloroplast-made boostervaccine to confer immunity against different polio virus serotypes. Plant Biotechnol J doi: 10.1111/pbi.12575.


Daniell H, Lin CS, Yu M, Chang WJ (2016A) Chloroplast genomes: diversity, evolution and applications in genetic engineering.Genome Biol DOI 10.1186/s13059-016-1004-2


Daniell H, Chan HT, Pasoreck EK (2016B) Vaccination through chloroplast genetics: Affordable protein drugs for the preventionand treatment of inherited or infectious diseases. Annual Review of Genetics, 50: in press


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Daniell H, Vivekananda J, Nielsen BL, Ye GN, Tewari KK, Sanford JC (1990) Transient foreign gene expression in chloroplasts ofcultured tobacco cells after biolistic delivery of chloroplast vectors. Proc Natl Acad Sci USA 87:88-92


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De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formationof insecticidal crystals. Nat Biotechnol 19:71-74

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Lakshmi PS, Verma D, Yang X, Lloyd B, Daniell H (2013) Low cost tuberculosis vaccine antigens in capsules: expression inchloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS One 8:e54708


Lee SB, Li B, Jin S, Daniell H (2011) Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 inchloroplasts to control viral and bacterial infections. Plant Biotechnol J 9:100-115


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León IR, Schwämmle V, Jensen ON, Sprenger RR (2013) Quantitative assessment of in-solution digestion efficiency identifiesoptimal protocols for unbiased protein analysis. Mol Cell Proteomics 12:2992-3005


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Pubmed: Author and Title https://plantphysiol.orgDownloaded on December 24, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Biotechnol J%5BTitle%5D%29 AND 11%5BVolume%5D AND 77%5BPagination%5D AND Kwon KC%2C Nityanandam R%2C New JS%2C Daniell H%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kwon KC, Nityanandam R, New JS, Daniell H (2013) Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells. Plant Biotechnol J 11:77-86

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kwon&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kwon&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Low cost tuberculosis vaccine antigens in capsules%3A expression in chloroplasts%2C bio%2Dencapsulation%2C stability and functional evaluation in vitro%2E%5BTitle%5D%29 AND Lakshmi PS%2C Verma D%2C Yang X%2C Lloyd B%2C Daniell H%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lakshmi&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lakshmi&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

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http://search.crossref.org/?page=1&rows=2&q=Lee SB, Li B, Jin S, Daniell H (2011) Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections. Plant Biotechnol J 9:100-115

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lee&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lee&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Transgenic Res%5BTitle%5D%29 AND 17%5BVolume%5D AND 1091%5BPagination%5D AND Lenzi P%2C Scotti N%2C Alagna F%2C Tornesello ML%2C Pompa A%2C Vitale A%2C De Stradis A%2C Monti L%2C Grillo S%2C Buonaguro FM%2C Maliga P%2C Cardi T%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lenzi P, Scotti N, Alagna F, Tornesello ML, Pompa A, Vitale A, De Stradis A, Monti L, Grillo S, Buonaguro FM, Maliga P, Cardi T (2008) Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco. Transgenic Res. 17:1091-1102.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lenzi&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lenzi&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Mol Cell Proteomics%5BTitle%5D%29 AND 12%5BVolume%5D AND 2992%5BPagination%5D AND Le�n IR%2C Schw�mmle V%2C Jensen ON%2C Sprenger RR%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Le�n IR, Schw�mmle V, Jensen ON, Sprenger RR (2013) Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics 12:2992-3005

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Le�n&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Le�n&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nucleic Acids Res%5BTitle%5D%29 AND 34%5BVolume%5D AND 3842%5BPagination%5D AND Lung B%2C Zemann A%2C Madej MJ%2C Schuelke M%2C Techritz S%2C Ruf S%2C Bock R%2C H�ttenhofer A%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lung B, Zemann A, Madej MJ, Schuelke M, Techritz S, Ruf S, Bock R, H�ttenhofer A (2006) Identification of small non-coding RNAs from mitochondria and chloroplasts. Nucleic Acids Res 34:3842-3852

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lung&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Expression of bar in the Plastid Genome Confers Herbicide Resistance&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lutz&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Plastid transformation of high-biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV-1) p24 antigen&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Plastid transformation of high-biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV-1) p24 antigen&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=McCabe&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Bioinformatics%5BTitle%5D%29 AND 26%5BVolume%5D AND 966%5BPagination%5D AND MacLean B%2C Tomazela DM%2C Shulman N%2C Chambers M%2C Finney GL%2C Frewen B%2C Kern R%2C Tabb DL%2C Liebler DC%2C MacCoss MJ%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Skyline: an open source document editor for creating and analyzing targeted proteomics experiments&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Skyline: an open source document editor for creating and analyzing targeted proteomics experiments&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=MacLean&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Biotechnol%5BTitle%5D%29 AND 145%5BVolume%5D AND 377%5BPagination%5D AND Madesis P%2C Osathanunkul M%2C Georgopoulou U%2C Gisby MF%2C Mudd EA%2C Nianiou I%2C Tsitoura P%2C Mavromara P%2C Tsaftaris A%2C Day A%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Madesis P, Osathanunkul M, Georgopoulou U, Gisby MF, Mudd EA, Nianiou I, Tsitoura P, Mavromara P, Tsaftaris A, Day A (2010) A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera. J Biotechnol 145:377-386

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http://scholar.google.com/scholar?as_q=A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Madesis&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Vaccine%5BTitle%5D%29 AND 30%5BVolume%5D AND 4225%5BPagination%5D AND Miyata T%2C Oshiro S%2C Harakuni T%2C Taira T%2C Matsuzaki G%2C Arakawa T%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Miyata T, Oshiro S, Harakuni T, Taira T, Matsuzaki G, Arakawa T (2012) Physicochemically stable cholera toxin B subunit pentamer created by peripheral molecular constraints imposed by de novo-introduced intersubunit disulfide crosslinks. Vaccine 30:4225-4232

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http://scholar.google.com/scholar?as_q=Physicochemically stable cholera toxin B subunit pentamer created by peripheral molecular constraints imposed by de novo-introduced intersubunit disulfide crosslinks&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Miyata&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Codon usage tabulated from international DNA sequence databases%3A status for the year 2000%2E%5BTitle%5D%29 AND Nakamura Y%2C Gojobori T%2C Ikemura T%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Codon usage tabulated from international DNA sequence databases: status for the year 2000.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Codon usage tabulated from international DNA sequence databases: status for the year 2000.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nakamura&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Cooperation between the chloroplast psbA 5%27%2Duntranslated region and coding region is important for translational initiation%3A the chloroplast translation machinery cannot read a human viral gene coding region%2E%5BTitle%5D%29 AND Nakamura M%2C Hibi Y%2C Okamoto T%2C Sugiura M%5BAuthor%5D&dopt=abstract


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Nakamura, M. and Sugiura, M (2007) Translation efficiencies of synonymous codons are not always correlated with codon usage intobacco chloroplasts. Plant J 49:128-134


Picotti P, Aebersold R (2012) Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions.Nat Methods. 9:555-566


Quesada-Vargas T, Ruiz ON, Daniell H (2005) Characterization of heterologous multigene operons in transgenic chloroplasts:transcription, processing, and translation. Plant Physiol 8:1746-1762


Ruhlman T, Verma D, Samson N, Daniell H (2010) The role of heterologous chloroplast sequence elements in transgeneintegration and expression. Plant Physiol 152:2088-2104


Savas JN, Stein BD, Wu CC, Yates JR 3rd (2011) Mass spectrometry accelerates membrane protein analysis. Trends Biochem Sci.36:388-96


Shenoy V, Kwon KC, Rathinasabapathy A, Lin S, Jin G, Song C, Shil P, Nair A, Qi Y, Li Q, Francis J, Katovich MJ, Daniell H, RaizadaMK (2014) Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuatespulmonary hypertension. Hypertension 64:1248-1259


Sherman A, Su J, Lin S, Wang X, Herzog RW, Daniell H (2014) Suppression of inhibitor formation against FVIII in a murine model ofhemophilia A by oral delivery of antigens bioencapsulated in plant cells. Blood 124:1659-1668


Shil PK, Kwon KC, Zhu P, Verma A, Daniell H, Li Q (2014) Oral delivery of ACE2/Ang-(1-7) bioencapsulated in plant cells protectsagainst experimental uveitis and autoimmune uveoretinitis. Mol Ther 22:2069-2082


Streng AS, de Boer D, Bouwman FG, Mariman EC, Scholten A, van Dieijen-Visser MP, Wodzig WK (2016) Development of atargeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T. JProteomics. 136:123-132


Surzycki R, Greenham K, Kitayama K, Dibal F, Wagner R, Rochaix JD, Ajam T, Surzycki S (2009) Factors effecting expression ofvaccines in microalgae. Biologicals. 37:133-138


Verma D, Moghimi B, LoDuca PA, Singh HD, Hoffman BE, Herzog RW, Daniell H (2010) Oral delivery of bioencapsulatedcoagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice. Proc Natl Acad Sci USA 107:7101-7106


Verma D, Samson NP, Koya V, Daniell H (2008) A protocol for expression of foreign genes in chloroplasts. Nat Protoc 3:739-758Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Waheed MT, Thönes N, Müller M, Hassan SW, Gottschamel J, Lössl E, Kaul HP, Lössl AG (2011a) Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plantsshow pleiotropic phenotypes. Plant Biotechnol J 9:651-660


http://search.crossref.org/?page=1&rows=2&q=Nakamura M, Hibi Y, Okamoto T, Sugiura M (2016) Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region. Plant J doi: 10.1111/tpj.13150.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nakamura&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nakamura&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant J%5BTitle%5D%29 AND 49%5BVolume%5D AND 128%5BPagination%5D AND Nakamura%2C M%2E and Sugiura%2C M%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Nakamura, M. and Sugiura, M (2007) Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts. Plant J 49:128-134

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nakamura,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nakamura,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nat Methods%5BTitle%5D%29 AND 9%5BVolume%5D AND 555%5BPagination%5D AND Picotti P%2C Aebersold R%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Picotti P, Aebersold R (2012) Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Methods. 9:555-566

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Picotti&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Picotti&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Physiol%5BTitle%5D%29 AND 8%5BVolume%5D AND 1746%5BPagination%5D AND Quesada%2DVargas T%2C Ruiz ON%2C Daniell H%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Quesada-Vargas T, Ruiz ON, Daniell H (2005) Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation. Plant Physiol 8:1746-1762

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Quesada-Vargas&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Quesada-Vargas&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Physiol%5BTitle%5D%29 AND 152%5BVolume%5D AND 2088%5BPagination%5D AND Ruhlman T%2C Verma D%2C Samson N%2C Daniell H%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ruhlman T, Verma D, Samson N, Daniell H (2010) The role of heterologous chloroplast sequence elements in transgene integration and expression. Plant Physiol 152:2088-2104

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ruhlman&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The role of heterologous chloroplast sequence elements in transgene integration and expression&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The role of heterologous chloroplast sequence elements in transgene integration and expression&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ruhlman&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Trends Biochem Sci%5BTitle%5D%29 AND 36%5BVolume%5D AND 388%5BPagination%5D AND Savas JN%2C Stein BD%2C Wu CC%2C Yates JR 3rd%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Savas JN, Stein BD, Wu CC, Yates JR 3rd (2011) Mass spectrometry accelerates membrane protein analysis. Trends Biochem Sci. 36:388-96

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Savas&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Mass spectrometry accelerates membrane protein analysis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Hypertension%5BTitle%5D%29 AND 64%5BVolume%5D AND 1248%5BPagination%5D AND Shenoy V%2C Kwon KC%2C Rathinasabapathy A%2C Lin S%2C Jin G%2C Song C%2C Shil P%2C Nair A%2C Qi Y%2C Li Q%2C Francis J%2C Katovich MJ%2C Daniell H%2C Raizada MK%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Shenoy V, Kwon KC, Rathinasabapathy A, Lin S, Jin G, Song C, Shil P, Nair A, Qi Y, Li Q, Francis J, Katovich MJ, Daniell H, Raizada MK (2014) Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension. Hypertension 64:1248-1259

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Shenoy&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shenoy&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Blood%5BTitle%5D%29 AND 124%5BVolume%5D AND 1659%5BPagination%5D AND Sherman A%2C Su J%2C Lin S%2C Wang X%2C Herzog RW%2C Daniell H%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sherman A, Su J, Lin S, Wang X, Herzog RW, Daniell H (2014) Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells. Blood 124:1659-1668

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http://scholar.google.com/scholar?as_q=Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sherman&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Mol Ther%5BTitle%5D%29 AND 22%5BVolume%5D AND 2069%5BPagination%5D AND Shil PK%2C Kwon KC%2C Zhu P%2C Verma A%2C Daniell H%2C Li Q%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Shil PK, Kwon KC, Zhu P, Verma A, Daniell H, Li Q (2014) Oral delivery of ACE2/Ang-(1-7) bioencapsulated in plant cells protects against experimental uveitis and autoimmune uveoretinitis. Mol Ther 22:2069-2082

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Proteomics%5BTitle%5D%29 AND 136%5BVolume%5D AND 123%5BPagination%5D AND Streng AS%2C de Boer D%2C Bouwman FG%2C Mariman EC%2C Scholten A%2C van Dieijen%2DVisser MP%2C Wodzig WK%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Streng AS, de Boer D, Bouwman FG, Mariman EC, Scholten A, van Dieijen-Visser MP, Wodzig WK (2016) Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T. J Proteomics. 136:123-132

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Streng&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Streng&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Biologicals%5BTitle%5D%29 AND 37%5BVolume%5D AND 133%5BPagination%5D AND Surzycki R%2C Greenham K%2C Kitayama K%2C Dibal F%2C Wagner R%2C Rochaix JD%2C Ajam T%2C Surzycki S%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Factors effecting expression of vaccines in microalgae&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Surzycki&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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Waheed MT, Thönes N, Müller M, Hassan SW, Razavi NM, Lössl E, Kaul HP, Lössl AG (2011b) Transplastomic expression of amodified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effectivesecond-generation vaccines. Transgenic Res 20:271-282


Wang X, Su J, Sherman A, Rogers GL, Liao G, Hoffman BE, Leong KW, Terhorst C, Daniell H, Herzog RW (2015a) Plant-based oraltolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells. Blood 125:2418-2427


Wang YP, Wei ZY, Zhong XF, Lin CJ, Cai YH, Ma J, Zhang YY, Liu YZ, Xing SC (2015b). Stable expression of basic fibroblast growthfactor in chloroplasts of tobacco. Int J Mol Sci 17:E19


Ye GN, Hajdukiewicz PTJ, Broyles D, Rodriquez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261-270


Yu CH, Dang Y, Zhou Z, Wu C, Zhao F, Sachs MS, Liu Y (2015) Codon usage influences the local rate of translation elongation toregulate co-translational protein folding. Mol Cell 59:744-754


Yukawa M, Kuroda H, Sugiura M (2007) A new in vitro translation system for non-radioactive assay from tobacco chloroplasts:effect of pre-mRNA processing on translation in vitro. Plant J 49:367-376.


Zoschke R, Barkan A (2015) Genome-wide analysis of thylakoid-bound ribosomes in maize reveals principles of cotranslationaltargeting to the thylakoid membrane. Proc Natl Acad Sci USA 112:E1678-87


Zoschke R, Watkins KP, Barkan A (2013) A rapid ribosome profiling method elucidates chloroplast ribosome behavior in vivo. PlantCell 25:2265-2275


Zou Z, Eibl C, Koop HU (2003) The stem-loop region of the tobacco psbA 5'UTR is an important determinant of mRNA stability andtranslation efficiency. Mol Genet Genomics. 269:340-349.



http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Biotechnol J%5BTitle%5D%29 AND 9%5BVolume%5D AND 651%5BPagination%5D AND Waheed MT%2C Th�nes N%2C M�ller M%2C Hassan SW%2C Gottschamel J%2C L�ssl E%2C Kaul HP%2C L�ssl AG%5BAuthor%5D&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Transgenic Res%5BTitle%5D%29 AND 20%5BVolume%5D AND 271%5BPagination%5D AND Waheed MT%2C Th�nes N%2C M�ller M%2C Hassan SW%2C Razavi NM%2C L�ssl E%2C Kaul HP%2C L�ssl AG%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Waheed MT, Th�nes N, M�ller M, Hassan SW, Razavi NM, L�ssl E, Kaul HP, L�ssl AG (2011b) Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines. Transgenic Res 20:271-282

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Waheed&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Waheed&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Blood%5BTitle%5D%29 AND 125%5BVolume%5D AND 2418%5BPagination%5D AND Wang X%2C Su J%2C Sherman A%2C Rogers GL%2C Liao G%2C Hoffman BE%2C Leong KW%2C Terhorst C%2C Daniell H%2C Herzog RW%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28%2E%5BTitle%5D%29 AND Wang YP%2C Wei ZY%2C Zhong XF%2C Lin CJ%2C Cai YH%2C Ma J%2C Zhang YY%2C Liu YZ%2C Xing SC%5BAuthor%5D&dopt=abstract

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http://search.crossref.org/?page=1&rows=2&q=Ye GN, Hajdukiewicz PTJ, Broyles D, Rodriquez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261-270

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ye&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ye&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Mol Cell%5BTitle%5D%29 AND 59%5BVolume%5D AND 744%5BPagination%5D AND Yu CH%2C Dang Y%2C Zhou Z%2C Wu C%2C Zhao F%2C Sachs MS%2C Liu Y%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yu&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zou&hl=en&c2coff=1

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new tools to study transgene expression in chloroplasts ...jul 27, 2016 · 3 52 introduction 53...

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