Spider Silk Expression in Alfalfa

Spider Silk Expression in
Alfalfa
Monica Rowan with Dr. Randy Lewis & Dr. Holly
Steinkraus- Molecular Biology, Dr. Robin Groose- Plant
Sciences
Molecular Biology and Plant Sciences
University of Wyoming
www.riverdeep.net/.../09/090301_spider.jhtml

Silk in spiders

Why Spider Silk?
Comparisons of Mechanical
Properties of Spider Silk
Material Strength (Nm -2) Elasticity (%) Energy to Break (Jkg -1)
Dragline Silk 4 x 10^9 35 4 x 10^5
KEVLAR 4 x 10^9 5 3 x 10^4
Rubber 1 x 10^6 600 8 x 10^4
Tendon 1 x 10^9 5 5 x 10^3__________
• Data derived from Gosline, J.M.; Dennv, M.W.; Demont, M.E. Nature 1984,
309,551

What can silk be used for?
‣ New textiles
‣ Improved shielding material that
could take the place of Kevlar in
bulletproof vests.
‣ Composite materials for building and
aerospace applications
‣ Fine sutures, artificial tendons and
ligaments

Why Plant Expression?
‣Low cost (10-50 fold
less than in E. coli).
‣Easy to scale up.
‣Agricultural
advances provide
for rapid harvesting
and processing.
http://www.wapa.gov/es/pubs/esb/1999/99Oct/at_clinton.htm

Plant Expression of Silk
• Genetic Engineering of
plants is well
established
• Adaptation as largescale
bioreactors for
commercially valuable
molecules
• Production of spider
silk has been hindered
because of lack of a
suitable large scale
production system
Tobacco
Arabidopsis
Potato
Alfalfa

Alfalfa (Medicago sativa L.)
‣ Perennial crop,
adaptable
‣ Open to transformation
‣ High protein content
- Up to 24% of dry weight
is protein
- If expressed at 1-2% of
TSP, yield of 218 kg of
silk protein/acre/year
- 2.5–12g/kg leaf tissue
‣ High MW proteins
http://www.ars.usda.gov/images/docs/3172_3356/Alfalfa%20Flower.jpg

Comparison of various expression systems
for the production of recombinant silk protein
under optimized conditions.
Expression
System
Protein
Concentration
Total Protein
Yield
Production
Time
Bacteria 100mg/L; 30,000L
fermentor
3kg/run
2-4 months
Goats
15g/L; 8L/day; with
a lactation of 150 d
18kg/goat/year
1-2 years
Alfalfa
1% of total soluble
protein, 10T/acre
assuming 5
cuttings/year
218kg/acre/year
4-5 years

Project Objectives
1) Insert a synthetic gene for
spider silk into the alfalfa
genome.
2) Analyze resultant plants to
determine gene copy number
and protein expression level.
3) Design an effective protocol
to purify spider silk proteins
from alfalfa.
4) Cross plants expressing silk
with a Wyoming adapted
cultivar.

Original silk constructs
‣Synthetic constructs based on N. clavipes sequence.
‣E. coli codon bias
‣MaSp 1 -> 16 repeats of 99 bp, each encoding:
GGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAA
GGX = 3 10 helix = elasticity (GA)n/(A)n = beta sheet = strength

Transformation of alfalfa
Selective + hormones: formation of calli
‣ A. tumefaciens: LBA4404
‣ Alfalfa: Regen S27
‣Transformed ~120 leaves/clone
‣Explants moved to soil: 30/clone
•kan
•ticar
•kinetin
•2,4-D
Selective – hormones: encourages root and shoot development

Parent Generation
‣MaSp1 + C #7 X WisFal
‣Analysis of F1 progeny
– Southern (DNA level)
– Western (Protein level)

F1 Southern Blot analysis
‣Digested alfalfa genomic DNA with the restriction enzyme NdeI.
‣Hybridized with a radioactively-labeled homologous probe.
‣ Examined images for the presence of a 2.3kb-hybridizing fragment,
which indicated the silk gene was incorporated into the plant genome.

Extraction of TSP’s from
MaSp1+C-transformed alfalfa
‣ Ground leaves in liquid
N 2
‣ Prepared crude extract
‣ Heat-treated soluble
extract; 90 o C, 10’
‣ Pelleted precipitated
proteins
‣ Examined by SDS-
PAGE

Western Blot Analyses
MW
G1
G3
B3
O1
WT
M4 (+)
75
50
61kD Spider Silk
Protein Band
AB α M 4 (MaSp1)

Summary of Southern/Western
results
‣105 F1 plants analyzed
- 74 positive on DNA level
- 4 positive on the protein expression
level
‣Consequently only 4% of the F1 plants
tested are expressing the silk protein.

WHY such low numbers?
‣Gene Silencing
– Multiple transgene copies
– Methylation of promoter region
‣Protein instability….protein itself
– No subcellular targeting

Improvements
‣ Change codon usage (increase yield):
- original constructs -> E. coli codon bias
- designed new constructs -> alfalfa codon bias
‣ Change vector (ER retention signal):
- original constructs -> pIBT110
- use new vector -> pCB301-KDEL

New binary vector
‣ pCB301-kan-CaMv-leb4sig-100xELP-cmyc-KDEL
100xELP
100xELP
(VPGXX) 100
KDEL
LB
nptIII (kanR)
cmyc tag
legumin
signal peptide
Met
CaMv 35S
promoter
DraIII
BamHI
XbaI
pCB301-kan 100 ELP
8359 bp
neo (kanR)
RB

Future Work
‣Collaboration with Medicago (Canada)
- New vectors
‣Transient Assay Development
‣Transformed leaves with new constructs
and analyze resultant plants.

Acknowledgements
Randy Lewis, Ph.D.
Holly Steinkraus, Ph.D.
Robin Groose, Ph.D.
Entire Lewis Lab
NSF EPSCoR for the opportunity

Questions?