systems for molecular farming · systems for molecular farming udo conrad, ipk gatersleben...
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![Page 1: Systems for Molecular Farming · Systems for Molecular Farming Udo Conrad, IPK Gatersleben Production of spider silk proteins in plants Production of recombinant antibodies in plant](https://reader035.vdocuments.us/reader035/viewer/2022062219/5f0caa367e708231d43687af/html5/thumbnails/1.jpg)
Systems for Molecular Farming
Udo Conrad, IPK Gatersleben
Production of spider silk proteins in plants
Production of recombinant antibodies in plant seeds
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costs per unit
market volume
therapeutic antibodies
fibers
Types of products in biofarming
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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
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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
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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.
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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
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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
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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
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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
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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
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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 )
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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
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Production system and further experiments
Proof for other promoters GABI
Proof for crop plants GABI
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Acknowledgements
Members of the phytoantibody group at IPK,
especially Jürgen Scheller and Michael Leps