Lecture 5: Biotechnology

and prokaryotes

•Bacteria and bacterial systems;•Manipulation of gene expression in prokaryotes (Chapt 6); •Large-scale purification of proteins from recombinant microorganisms (Chapt 16)

Biotechnology and prokaryotes

•“germs”•Prokaryotes are ubiquitous•Wide spectrum of species, types, characteristics•“Relatively simple” systems, for manipulation•Biology characterized•Genetics characterized•[Molecular biology characterized]•Genomics, bioinformatics, systems•Industrialization, [weapon-ization]•Two-edged blade: “anti- and pro-”•Effects on humans, other organisms, environment

•[medical (pathogen), therapeutic (food)]…•Less legal, ethical, moral issues

Phylogeny and range of

prokaryotes

Phylogenic branches

•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

Shapes and “multicellular”-ism

•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

•quorum•fluorescence•biofilms

•‘differentiation’- Myxococcus xanthus

•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

Optimum growth temperature

Structure of a type of bacterium

Types of bacteria: gram positive vs

gram negative

Characterization of bacteria: Gram stain•Hans Christian Gram, 1882•Crystal violet -> methylene blue

•wikipedia

Types of bacteria: gram positive vs gram negative

•Gram positive:•Thick cell wall, peptidoglycan•Examples: Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, Clostridium

•Gram negative:•Cell wall, lipopolysaccharide (also, LPS or endotoxin layer) plus [less] peptidoglycan•Examples: Escherichia coli, Salmonella, Pseudomonas, Legionella

wikipedia

Bacillus types and biotechnology

Gram positive:•Contrast B. anthracis vs. B. thuringiensis vs B. subtilis•Spores vs crystals•Ag biotech

•http://www.scq.ubc.ca/wp-content/uploads/2006/08/GM-crop.gif•http://images.google.com/imgres?imgurl=http://www.magma.ca/~scimat/B_thur16.jpg&imgrefurl=http://www.magma.ca/~scimat/B_thurin.htm&h=324&w=340&sz=23&tbnid=4A1w35t4YFto8M:&tbnh=113&tbnw=119&prev=/images%3Fq%3Dbacillus%2Bthuringiensis%26um%3D1&start=1&sa=X&oi=images&ct=image&cd=1•wikipedia

Bacillus and biotechnology

Gram positive:•B. thuringiensis•Spores vs crystals•Ag biotech

•http://www.scq.ubc.ca/wp-content/uploads/2006/08/GM-crop.gif•http://images.google.com/imgres?imgurl=http://www.magma.ca/~scimat/B_thur16.jpg&imgrefurl=http://www.magma.ca/~scimat/B_thurin.htm&h=324&w=340&sz=23&tbnid=4A1w35t4YFto8M:&tbnh=113&tbnw=119&prev=/images%3Fq%3Dbacillus%2Bthuringiensis%26um%3D1&start=1&sa=X&oi=images&ct=image&cd=1•wikipedia

Wall-less bacteria

•Wall-less bacteria•Smallest, simplest free-living self-replicating organisms; still, complex•Reduced genome species•~100 species of bacteria and archaea•Membrane ~30% lipid and 70% protein•25-30% cholesterol, comparable to eukaryotes•[other bacteria do not have cholesterol or sterols in PM]•Example: Mycoplasma pneumoniae, Thermoplasma acidophilum

•L-form or cell wall deficient bacteria•Chronic disease? [rheumatoid arthritis, Chronic Fatigue Syndrome, Lyme disease]

•[http://bacteriality.com/2007/08/18/history/]

Life on the Edge

•Deinococcus radiodurans (eubacteria)•Survives 5 Mrads

•DNA highly damaged at 1.7 Mrads; 1,000 to 2,000 DNA fgmts repaired in 24 hrs•RE map; Four genome elements, circular <-> linear large

http://www.ornl.gov/sci/techresources/Human_Genome/publicat/microbial/image3.html

Life on the Edge

•Science Daily (Source: NASA, +), 4/4/00 “Weird Life”•The limits of life on Earth are much broader than previously thought. •Examples of life at extreme conditions include:

•Highest radiation: 5 million rads-- Deinococcus radiodurans (eubacteria)•Hottest: 235.4 F (113 C)-- bacteria from deep sea vents•Coldest: 5 F (-15 C)-- microalgae in Antarctic rocks•Deepest: Two miles underground in rocks-- bacteria•Most acidic: pH 0-- Unclassified organisms growing on gypsum in caves•Basic: pH 9.0; 12% salt-- bacteria, Soap Lake, Southeast WA•Saltiest: 30 percent salt environment-- bacteria•Deepest and Highest pressure: 1200 atm-- at bottom of Marianas Trench (ocean)•[Farthest: Moon-- Streptococcus mitus (from human source) from Surveyor III camera after three years unprotected on lunar surface]

Varied growth rates,

oxygen needs

•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf

•20 min: E. coli•24 hrs: M. tuberculosis

Manipulating bacteria

•http://www.mansfield.ohio-state.edu/~sabedon/black06.htm

Bacterial growth

•http://www-micro.msb.le.ac.uk/labwork/bact/bact1.htm

Bacterial growth: quantify

Natural selection and

growth

•Thomas Malthus. 1798, “Essay on the Principle of Population”•“population would outstrip food supply”

•Charles Darwin. 1859, “The Origin of Species” •“an application of doctrines of Malthus”

Natural selection and

growth: How?

•Glucose- depleted•Fructose- not available•Maltose- not available•Lactose- available

Microbial genetics and

molecular biology

•Genes and pathways are under strict regulatory control

Microbial genetics and molecular biotechnology

•Control timing of expression of foreign gene product•Lac promoter

•Repressed by lac repressor•Derepressed by lactose and IPTG•lacUV5 -nuc change in -10 region•Stronger promoter

•Trp promoter•Repressed by trp repressor•Derepressed by trp and indoleacrylic acid•“leaky”

hybrid promoter: tac

•Overexpression of foreign gene product•eg, lac repressor protein:•~10 molecules per cell•‘need’ ~1 umol for biochem studies•1 umol = 6 x1017 molecules•Or 6 x1016 cells•E. coli at stationary phase has•~4 x108 cells/mL•*if* 100% recovery, need 150,000 L

•tac promoter•Hybrid of trp and lac promoters•Matches consensus for E. coli RNAP seq•HAdeBoer, et al. 83. PNAS 80:21

hybrid promoter: tac[not always straight-

forward]

•tac promoter•tacI, tacII

•lacUV5 -nuc change in -10 region •Relative strengths

•tac/lac 11.8x; tac/trp 3.5x•Strain dependent•HAdeBoer, et al. 83. PNAS 80:21

Bacteriophage genetics

Bacteriophage genetics and molecular biology

•Lambda promoter•Temperature sensitive promoter•Repressed unless temperature raised•Overexpression of foreign gene product

•incompatible or lethal/deleterous

dual promoters: lac and T7

•Repressed unless inducer is present•Overexpression of foreign gene product

•incompatible or lethal/deleterous

Plasmids

•JLederberg, 1952. Physiol. Rev. 32: 403 -”plasmid”•Circular, extrachromosomal double-stranded DNA•Size: 1 kb to 400 kb•Number of copies: 1-2 to 20s to 100s ~ori

•wikipedia

Plasmids

•Conjugation- horizontal gene transfer•vs ‘episome’ (plasmid that can integrate into chromosomal DNA)

•Size: 1 kb to 400 kb•Number: 1-2 to 20s to 100s ~ori wikipedia

Overexpression strategy: construction of hybrid vector

•Have: pPLc2833•Strong promoter pL, selectable marker, MCS

•Have: pKN402 ori gives increased copy number 5-10x•Temperature-dependent copy number

•Construct: hybrid pCP3•pL promoter and ApR gene with high-copy ori

Overexpression strategy: plasmid number increase

•Have: pPLc2833•Strong promoter pL, selectable marker, MCS

•Have: pKN402 ori gives increased copy number 5-10x•Temperature-dependent copy number

•Construct: hybrid pCP3•pL promoter and ApR gene with high-copy ori

Large-scale prep considerations

•IPTG and other derepressing chemicals can become expensive•Temperature-dependent shifts: time and energy for large cultures•Reconfigure pL with trp promoter•Grow in molasses and casein hydrolysate, low in free trp; derepress with tryptone [crude extract]•ex, -galactosidase and citrate synthase genes-> overproduced to 21% and 24% of cellular proteins

Other bacterial hosts

•Genetics and molecular biology not as well-developed as E. coli•Using E. coli system in other gram negatives•(Nm is neomycin resistance; S1 is R. meliloti ribo protein gene)

Universal gram negative

vector

•70 bp fgmt from Tn-5 Terminal IR•Low copy-number broad-host-range plasmid•E. coli, Alcaligenes sp, Enterobacter cloacae, Klebsiella pneumoniae,•Pseudomonas stutzeri, Pseudomonas fluorescens, Serratia marcescens

Modifications for human

consumption

•Lactic acid metabolizing bacteria, eg Lactococcus spp•Dairy products- cheese and yogurt•Desirable to increase yields, add to quality of food•Undesirable to alter production process, product palatability, appearance, etc.•Cannot add chemical inducers or temperature effects to processing•Consensus constitutive promoter--> synthetic constitutive promoters•Tested 36 constructs•Most active ~7,000x stronger than least active•If -10 or -35 regions intact, ~400x in strength

Fusion proteins

•Not all heterologously expressed proteins are expressed•Not all heterologously expressed proteins are expressed ‘enough’•Not all heterologously expressed proteins are stable as expressed•Engineer a fusion protein that has a partial structure of a ‘host-native’•Increase expression•Extend half-life•Requires correct reading frame•Above, proteolytic cleavage by blood coagulation factor Xa

•Val is N-term of desired protein•Based on existing biology….

Blood clotting

Post-translational modifications

•Extend half-life•Address protein delivery•Stabilize structure•Enhance biological activity

Vector for expression fused protein

•5’-terminal segment of ompF gene for producing antigen•Directs synthesis of outer membrane protein and part of -galactosidase•Omp provides signals for txn and tnl, and secretion of product•lacZ functional, as a reporter for in-frame•“tribrid” has all three products’ characteristics

Purification protocol

enhancement

•Efficient protocols for purification of product

Fusion construct example

•Product is a secretion fusion protein•Fusion is marker peptide plus interleukin-2•Marker protein serves to extend half-life and to enable rapid purification•One-step purification via immunoaffinity chromatography•Small marker peptide does not stress host resources•For FDA, removal specifically with bovine intestinal enterokinase, a protease

Fusion protein purification

Column chromatography/HPLC

“Recombinant DNA era” protein purification

•DNFrick and MJBessman JBC 270: 1529 (95) •Over-expression and purification of the Orf257 protein: SDS-PAGE and PAGE

A1) markers; A2 and 3) -/+IPTG;A 4 and 5) 2 and 6 ug Fraction VB1) markers; B2 and 3) 2 and 6 ug Fraction V

•Protocol:1. DEAE-Sepharose2. Streptomycin-SO4 fractionation3. Sephadex G-100 chromatography

Rapid screening methods:

bacteriophage

•Large libraries of recombinants- How to find clone of interest?•ex., cDNA library with 5 x1010 clones, proteins which are rarely occurring cDNAs

•Can fuse with a surface protein gene of either a filamentous phage or bacterium•After txn and tnl, fusion is expressed on the surface of the organism•Can screen with antibody

Rapid screening methods: bacterium

•Large libraries of recombinants- How to find clone of interest?•ex., cDNA library with 5 x1010 clones, proteins which are rarely occurring cDNAs

•Can fuse with a surface protein gene of either a filamentous phage or bacterium•After txn and tnl, fusion is expressed on the surface of the organism•Can screen with antibody

Using probes: practice, alternatives and updates(surface display)

Overexpression of foreign product

•Increasing plasmid copy number sometimes reduces yield of product•Cell resources diverted to production of other plasmid-encoded products•Creation of tandem arrays of the product•In-frame expression of multiple copies•Each copy has own signals•Copy number limits yield as well; unstable inserts

Translation expression vectors

•Requires more than regulated strong promoter•Efficiency of translation and stability of nascent peptide•Prokaryotes: proteins are synthesized at different efficiencies, up to 100x•One aspect- translational initiation signal or ribosome-binding site•If mRNA has hairpin, can disrupt•Above GGGGG is rbs and AUG is start

Construction of a

translation efficient vector

•ApR marker•tac promoter•lacZ ribosome-binding site•ATG start site, 8 nucs downstream from rbs•Two txn terminators T1 and T2 from lambda

Translation efficiency

•Different tRNA usage•AGG, AGA, AUA, CUA and CGA are least-used codons in E. coli•Engineer strains to have different pools•ex., h2 protein, a peanut allergen, overproduced 100x than in conventional strains

Posttranslational

stabilization

•Half-life ranges from few minutes to hours•Posttranslational modification of amino acids, •Including addition of single amino acid to N-term•Certain strings of AAc internally can increase proteolytic degradation

•PEST, rich in pro, glu, ser, thr•[change these sequences?]

Posttranslational problems: folding

•Insoluble proteins, esp when overexpressed•Non-biologically active•Fusion with 11.7 kDa thioredoxin allows target protein up to 40% of cellular proteins•Above, trp controls cI to shut target expression; no trp-> no cI -> target expression

Peptide folding aid

•Overproduction of E. coli disulfide bond-forming protein (DsbC) on •Synthesis of active human tissue plasminogen activator•Normal levels DsbC vs overproduction of DsbC with overproduction of tPA•tPA addressed to periplasm

Misc factors

•Oxygen limitation: aerobic, anaerobic, facultative anaerobic•Most microbes used for overproduction require oxygen for growth•Oxygen has limited solubility•Cell density can outstrip oxygen availability, leading to stationary phase•(oxygen is a resource, just as nutrients)•Esp in stationary phase, where degradative proteins are produced

•Protease-deficient strains•Develop strains that are deficient in degradative proteins•E. coli has >25 different proteases•Some are for house-keeping functions, eg removing abnormal and defective proteins•Most engineered strains are slow growers•One strain, rpoH- (heat shock synthesis sigma factor) and a gene for a protease-, secreted •Proteins with a 36x greater SA than from WT cells (indicates decrease in proteolysis)

•Bacterial hemoglobin•Some microbes, strains of Vitreoscilla -obligate aerobe-, live in oxygen-poor environments•For their oxygen needs, synthesize hemoglobin-like protein•Raises effective intracellular oxygen•Cloned into E. coli, more effective metabolism: higher levels protein synthesis, proton pumping,

higher ATP production rate, higher ATP concentration

Strategy: integration into chromosome

•Plasmids impose metabolic loads•Energy for replication, txn, tnl•Leads to unstable inserts, plasmids; slow growing

Chromosomal integration and expression

•B. subtilis•E. coli plasmid with B. amyloliquefaciens -amylase•Protocol to integrate into different predetermined sites on chromosome•At nonessential sites

Genome integration

•B. subtilis•E. coli plasmid with B. amyloliquefaciens -amylase•Protocol to integrate into different predetermined sites on chromosome•At nonessential sites

Removal selectable marker genes

•Selectable marker required for lab protocols•Release of antibiotics-resistance into environment not good•Cre is on a second plasmid in same cell; IPTG induced•Excision of marker

Increasing secretion

•Addressing: signal peptide•Stability depends on location of product:•Proinsulin is 10x more stable if inserted into membrane

Increasing secretion example: not simple

•Interleukin-2 fused with E. coli maltose-binding protein signal peptide•Requires a part of the MBP gene as well

Other strategies for secretion

•L-form bacteria are variants that lack cell wall•Arise via spontaneous mutation or treatment

•Penicillin- inhibits final step in cell wall formation•Lysozyme- hydrolyzes cell wall saccharide bonds

•Can transform with engineered vector:•L-form P. mirabilis•E. coli alk p’ase leader peptide/ Leu-Gly linker/Pro7/T. vulgaris carboxypeptidase/ txn term signal

•Carboxypeptidase secreted maximally after 36 hrs fermentation

Metabolic loads

•Overexpression of foreign protein

Metabolic loads, example

Lecture 5 (cont)

•Chapt 16•Large-scale production of proteins from recombinant microorganisms•Technical considerations

Generalized scheme for lg-sc fermentation process

Microbial growth

•Batch, fed-batch (staged addn of nutrients), continuous

Bacterial growth

Bioreactors

Two-stage fermentation in

tandem

•Temperature-dependent induction of a protein product

Fermentation vessels

Fermentation vessels

Fermentation vessels

Two-stage fermentation, one vat

•Tripartite fusion protein AGgal•Encodes five IgG binding domains of S. aureus protein A plus two IgG binding domains from S. str G148 protein G plus B-galactosidase E. coli•Driven with Lambda promoter•5 L culture with Ap and Kn seeds 600 L sans both•In 600 L, temp shift 30 to 40

•Loses 50% plasmids after 4 hrs•After 4 hr, yield at ~20% dry wgt of total biomass

•Saves on space, antibiotics, time (did not have to integrate into chromosome)

Batch vs fed-batch

fermentation

Batch vs fed-batch

fermentation

Harvesting cells: Bucket centrifuge

Harvesting cells: Sharples centrifuge

Harvesting: continuous

membrane filtration

Cell disruption

•Wet milling•High-pressure homogenization•Impingement•[sonication]•[enzymatic]

Downstream processing

•After disruption,•Cell debris removal by centrifugation or membrane microfiltration dialysis•One membrane, cut-off 10 kDa

Downstream processing

•After disruption,•Cell debris removal by centrifugation or membrane microfiltration dialysis•Two membranes, differential filtration