Systems for Molecular Farming

Udo Conrad, IPK Gatersleben

Production of spider silk proteins in plants

Production of recombinant antibodies in plant seeds

costs per unit

market volume

therapeutic antibodies

fibers

Types of products in biofarming

Spider silk proteins in transgenic plants

∼30

∼20

∼15

∼10

∼60∼40

∼90∼70

∼130

prot

ein

ladd

er(k

Da

)

potato

standardsSpider silkmonomers

Production of transgenic plants

Biochemical analysis and purification by heat and salt precipitaton

c-myc tagfibroin-spidroin1 hybrid

spidroin1

94.2 kDa1189 aa

ER retention(KDEL)

LeB4signal peptide

SO1 xELP

ELPelastin-like polypeptide

Expression of spidroin-ELP-fusion proteinsin the ER of transgenic pflants

Properties of temperature sensitive peptides:

ELP - Elastin Like PolypeptideOligomeres of the pentapeptide -Val-Pro-Gly-Xaa-Gly-

unsoluble under Tt (inverse transition temperature)

unsoluble above Tt (inverse transition temperature)

reversible desolvation, aggregation and prezipitation

2-3°C

reversible

! ELP´s have high homology to the spider silk gene Flag

37

75

50

100

150

2515

10

solu

ble

heat

heat

-20x

con

supe

rnat

ant

prot

ein

ladd

er(k

Da)

1 2 3 4 5

heat

+sal

t

resu

sp. p

elle

t

heat

+sal

t

Purification of spider silk-ELP fusion proteins from transgenic plants

Spidroin-ELP-fusions could be purifiedby addition of salt and by heatto 95% purity.

Fruit water

Potato

Grater

Starch

Fibers(Hemicellulose)

Heat

Soluble sub. Pülpe

Fractioningin wet-process

Protein

potato - spider silk proteinsas by-product ofstarch production

Filtration

Spider silk

Pülpe

Retinate

Filtrate

Production form

VKVH

single chain Fv antibodies:

specific scFv with high affinity available fromphage display librariesaccumulation in different compartments of plant cells andin different organs to high concentrations

Expression of recombinant antibodies in transgenic plants

Expression of scFv-ELP fusions in plant seeds

USP SP scFv* tag1 100xELP tag2 Term

KDEL

5´ 3´

Production of transgenic tobacco plants

*anti-fungizide scFv

Accumulation of ELP-scFv fusions in plant seeds to highconcentrations

Coomassie stain Silver stain

WT UB UB UB-ELP M WT UB UB-ELP M

scFv-ELPfusion protein

Accumulation of ELP-scFv fusions in tobacco seeds to high concentrations

~17% TSP

~20% TSP

~15% TSP

914UB-ELP

min. max. meanscFv expressingplants

Kan-resistentplants

construct

ScFv-ELP fusion proteins from seeds are highly active in ELISA

1 2 3 4 5 6

SNN

UB 64/4 (0.3% TSP)UB~ELP 6 (20% TSP)

0,423

1,88

0,19

0

0,5

1

1,5

2

2,5

3SNNUB 64/4UB~ELP 6

OD 405

Log (1/dilution extract )

ScFv-ELP fusion proteins and scFv show identicalbinding behaviour in competitive ELISA

0

0,2

0,4

0,6

0,8

1

1,2

0 M 1x10-7M 1x10-6M 1x10-5M 1x10-4M

UB_64/4UB~ELP 6

OD 405

Concentration of free antigen

Production system and further experiments

Proof for other promoters GABI

Proof for crop plants GABI

Acknowledgements

Members of the phytoantibody group at IPK,

especially Jürgen Scheller and Michael Leps