nicotiana benthamiana plants stably expressing the...

Production of alpha-1 antitrypsin in Nicotiana benthamiana plants stably

expressing the mammalian sialylation pathway

ALEXANDRA CASTILHO

Laura Bassi Centres of Expertise - PlantBioP

Dep. of Applied Genetics and Cell Biology Dep. of Chemistry

PBVAB, Lausanne 2015

http://www.w-fforte.at/index.php?id=1

PROTEIN N-GLYCOSYLATION N-linked glycosylation, is the attachment of an oligosaccharide known as glycan to a particular asparagine residue in a protein • most frequent post-translational modification

• important for:

– folding – stability – in vivo half-life – biological activity


GLYCO-ENGINEERING N. BENTHAMIANA


Plants Human

WT ΔXF ΔXF+GalT ΔXF+Sia

RNAi XylT/FucT Stable STGalT Transient Sia (6 genes)

ENGINEERING GLYCOSYLATION PATHWAY

ManNAc ManNAc-6-P

Neu5Ac-9-P

GNE

NANS

UDP-GlcNAc GNE Cytosol

NANP

Neu5Ac

Nucleus

CMP-Neu5Ac CMAS

CST

NeuAc

Galactose Mannose

GlcNAc

CMP-Neu5Ac

Asn GalT

ST

N-Glycosylation

Golgi

Asn

Asn


∆XTFT

GnTIV GnTV GE UGT GalNAcT2 GNE CMAS CST NANS ST ST6 GalNAc4

ST3GalI EPO GalT C1GalT1

N- and O-GLYCOENGINEERING OF EPO

Erythropoietin


http://www.clker.com/clipart-43240.html

















pPZp-RCS2

pSAT5

pSAT1 pSAT3

pSAT6 pSAT7

pSAT4

Individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple proteins from a single vector

MULTIPLE GENE ASSEMBLY


Target gene P T rare cutter rare cutter

AgeI NotI MCS

Assembly of multigene

Replacement of expression cassettes

pSATn

SAT1= octopine synthase promoter and terminator SAT2= nopalin2 synthase promoter and terminator SAT3=manopine synthase promoter and terminator SAT4=2x35S promoter and 35S terminator SAT6= rubisco small unit promoter and terminator SAT7=actine promoter and agropine synthase terminator

MODULAR SERIES OF PLASMIDS FOR MULTIPLE GENE EXPRESSION

IN PLANTS


GNE NANS AscI

ocsP ocsT 2x35S 35ST CMAS 2x35S 35ST

AscI LB RB I- SceI PI-PspI

pPZP-RCS2 Res

AscI I- SceI I- SceI PI-pspI PI-pspI

Cyt

opla

sm g

enes

pG371

ST CST AscI AscI

ocsP ocsT I- SceI

masP masT STGalT actP agsT

AscI LB RB I- SceI PI-PspI

pPZP-RCS2 Res

I- SceI PI-pspI PI-pspI

Gol

gi g

enes

MULTIPLE CASSETTE CONSTRUCTS

SAT1GNE SAT4NANS SAT4CMAS 2mEpsps

Ce144

SAT1basta SAT1ST SAT3CST SAT7GT

LB

Gb371


ManNAc ManNAc-6-P GNE NANS UDP-GlcNAc GNE Neu5Ac CMP-Neu5Ac CMAS

NANS CMAS 2mEpsps RB LB

Ce144 GNEMUT

CMAS: LB RB

ha P35S CMAST KanR g7T

mc GNE: LB RB

P35S GNE KanR g7T

NANS: LB RB

P35S NANS KanR g7T mc

mc

[CMP-Neu5Ac] pmol/mg

6.5

37.9

-

GNEMUT

Feedback inibition of GNE


SYNTHESIS OF SIALIC ACID IN PLANTA

EXPRESSION PLATFORM FOR IN PLANTA SIALYLATION

Ce144 Gb371 x

Ce144xGb371

Ce144+Gb371

Individual transformants Co-transformants


NANS CMAS 2mEpsps RB LB

Ce144 GNEMUT

GalT CST ST basta RB LB

Gb371

Gb371 Ce144 XTFT Ce144xGb371

PLANT EXPRESSION PLATFORMS FOR PROTEIN SIALYLATION


WT ΔXF GalT Sia

IN SUMMARY: EXPRESSION PLATFORMS

New


ALPHA-1 ANTITRYPSIN (A1AT) TRANSIENT EXPRESSED IN NICOTIANA BENTHAMIANA (POSTER 103)

Tnos LB Pnos Kan R PAct2 RB Tnos MP a SP TVCV - RdRp

Tnos LB Pnos Kan R PAct2 Tnos MP MP SP TVCV - RdRp GOI

magnICON® viral-based vectors for expression A1AT

RCL

358M

From David S. Goodsell and the RCSB PDB.

A1AT

Elastase


• A1AT deficiency causes pulmonary emphysema or COPD (chronic obstructive pulmonary disease)

• Therapeutic A1AT is currently purified from

pooled human serum • Protein products can cost up to $100,000 a

year per patient

70

55

40

TSP IF P

TSP: Total soluble proteins IF: intercellular fluid P: purified protein

Western Blot: anti-A1AT

6mg/g

PLANT-DERIVED A1AT

358M

Protein is cleaved near the centre of reactive loop typical of non-targeted serine proteases PBVAB, Lausanne 2015

TSP

A1AT IS PROCESSED IN VITRO BY INTERACTIONS

WITH PLANT SERINE PROTEASES.

serum plant


PLANT-DERIVED A1AT HAS PAUCIMANNOSIDIC

GLYCANS

WT XT/FT WT ΔXF

GnGnXF GnGn

MMXF MM

HEXO

WT ΔXF

GnGnXF GnGn


HEXOSAMINIDASE-3 IS RESPONSIBLE FOR

PAUCIMANNOSIDIC GLYCANS IN A1AT


SIALYLATION PREVENTS PAUCIMANNOSIDIC

GLYCANS IN A1AT

NANS CMAS Epsps RB LB

Ce144 GNEMUT

GalT CST ST basta RB LB

Gb371

CMAS: LB RB

ha P35S CMAST KanR Pnos Tnos g7T

CST: LB RB

P35S CST KanR Pnos Tnos g7T mc

STGalT: LB RB

P35S GalT KanR Pnos Tnos g7T CTSST

ST: LB RB

P35S ST KanR Pnos Tnos mc T35S

mc GNE: LB RB

P35S GNE KanR Pnos Tnos g7T

NANS: LB RB

P35S NANS KanR Pnos Tnos g7T mc

mc

TRANSIENT CO-EXPRESSION STABLE CO-EXPRESSION

- + Sia

c

TRANSIENT SIALYLATION STABLE SIALYLATION


70

55

40

TSP IF P

TSP: Total soluble proteins IF: intercellular fluid P: purified protein

Western Blot: anti-A1AT

PLANT-DERIVED A1AT IS ACTIVE

CLSM

merged ER-marker A1AT-GFP

A1AT+PPE

PPE

+PPE -PPE


IN SUMMARY

β-N-Acetylhexosaminidase 3 is responsible for the unusual Paucimannosidic N-Glycans on secreted A1AT

which can be avoided by deficiency of hexosaminidase 3 or sialylation.

Full-length A1AT accounts for about 40 % of the recombinantly expressed protein and is biologically active.

The majority of A1AT is produced with truncations at the C-terminus that destroys the reactive centre loop and

thereby abolishes the inhibitory activity of the protein.

A1AT is efficiently expressed in N. benthamiana : 6 mg of recombinant protein per gram of leaf material


ACKNOWLEDGMENTS

Herta Steinkellner Richard Strasser

Pia Gattinger Somanath Kallolimath

Thomas Hackl Lukas Mach

Andreas Loos

Hanna Weindorfer

Friedrich Altmann Clemens Gruber

Markus Windwarder

Chemistry Dep. Dep. of Applied Genetics and Cell Biology

Laura Bassi Centres of Expertise - PlantBioP

Victor Klimyuk Stefan Werner Carola Engler

http://www.chemie.boku.ac.at/134.html?&L=

http://www.dagz.boku.ac.at/dagz.html

http://www.w-fforte.at/index.php?id=1

nicotiana benthamiana plants stably expressing the...

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