Tailor-made biomaterials with tunable functionality are crucial for various applications, ranging from high performance fibers to tissue engineering. Our goal is to bio-engineer a novel family of spider silk biopolymers by taking control of chemistry, composition, and size to mimic properties of native spider silk. Since spiders are hard to farm like silkworms in high densities due to their carnivorous nature, we cannot produce large amounts of spider silk from farm-raised spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk for commercial applications. The relevant genes of spiders have been cloned and inserted into several different organisms, such as E. coli, goat and silkworm, to make spider silks. However, producing large-scale truly spider-like silk is still a big challenge due to the small protein size, low yield and low water solubility of bio-synthetic spider silk. The current study reports our recent progress in bio-synthetic production of spider silk protein in E. coli. We have constructed a plasmid vector with spider silk protein genes. With media optimization and protein induction studies, we have developed a fermentation process, which can express spider silk proteins in E. coli in the level at or above 0.5g/L.

1. PROTEIN PRODUCTION USING BIOREACTORS
Figure 2. Production of chimeric spider silks at (A) 5L (B) 20L (C) 100L volumes. Note: 1L bottle to scale
Funding: Utah Science Technology and Research (USTAR), the
Department of Energy (DOE), the National Science Foundation (NSF), and
the Department of Biology.
ACKNOWLEDGEMENTS
Tailor-made biomaterials with tunable functionality are crucial for
various applications, ranging from high performance fibers to tissue
engineering. Our goal is to bio-engineer a novel family of spider silk
biopolymers by taking control of chemistry, composition, and size to
mimic properties of native spider silk. Since spiders are hard to farm
like silkworms in high densities due to their carnivorous nature, we
cannot produce large amounts of spider silk from farm-raised spiders.
Genetic engineering is an alternative approach to produce large
quantities of spider silk for commercial applications. The relevant
genes of spiders have been cloned and inserted into several different
organisms, such as E. coli, goat and silkworm, to make spider silks.
However, producing large-scale truly spider-like silk is still a big
challenge due to the small protein size, low yield and low water
solubility of bio-synthetic spider silk. The current study reports our
recent progress in bio-synthetic production of spider silk protein in E.
coli. We have constructed a plasmid vector with spider silk protein
genes. With media optimization and protein induction studies, we have
developed a fermentation process, which can express spider silk
proteins in E. coli in the level at or above 0.5g/L.
ABSTRACT
Figure 1. Schematic overview to obtain chimeric silk proteins as a tool to expand their functional
features.(Top) The golden orb weaving spider Nephila clavipes produces the major ampullate 2 protein that
confers strength and elasticity to the fiber; whereas the flagelliform silk, making up the highly extensible
spiral capture, confers elasticity. The main secondary structures adopted by these sequences are illustrated.
(Bottom) The primary sequences of the consensus repeats of both proteins are depicted.
OVERVIEW
Large Scale Production of Spider Silk Protein in E. coli
Paula F. Oliveira, Sreevidhya Tarakkad Krishnaji, R. Chase Spencer, Jordan Wanlass, Dong Chen, Matthew Sims,
Michael Hinman, Justin Jones and Randolph V. Lewis
Department of Biology, Utah State University, UT 84341
BA C
MOLECULAR CHARACTERISTICS OF CHIMERIC SILKS
Protein
Code
Sequence Mol. Wt.
[kDa]
FlYS GPGGPGGY(GPGGSGPGGY)3GPGGSGPSGPGSAAAAAAAAG
P(GGYGPGGSGP)5GPGSAAAAAAAAGPGGYGP(GGSGPGGYG
P)2GGSGPSGPGSAAAAAAAAGP(GGYGPGGSGP)6SGPGSAAA
AAAAAGP(GGYGPGGSGP)8SGPGSAAAAAAAA
30.3
(FlYS) 3 3X FlYS 82.7
(FlYS)4 4X FlYS 108.9
RESULTS
RESULTS
CONCLUSIONS
Construction of expression vectors
pET19k cloning vector was generated by modifying pET19b vector
(Novagen; San Diego, CA) in which the ampicillin resistance gene was
replaced with the kanamycin resistance. In order to increase expression
levels of existing recombinant spider silks that are rich in glycine or proline,
we developed pET-SX from pET19k by incorporating the gene that results in
the expression of serine hydroxyl methyl transferase (SHMT) which converts
the amino acid serine to glycine and sequences that produce additional
tRNAs for glycine and proline.
Considerations for high-level expression in fermenter cultures
All protein expressions were carried out from freshly transformed E.coli. A
streak of colonies were picked and allowed to grow for 8h at 37°C, and
expanded overnight at 30°C to 5-8% starting volume to be inoculated into
the fermenter vessel. The specific growth rate of E. coli needs to be carefully
controlled to achieve a high cell density. The exponential feed line was
started at OD~4 to keep up with the growth. We also presumed that the
degree of the nutrient (mainly, glucose and amino acids) starvation at higher
ODs would significantly affect the variation of plasmid copy number in
recombinant cell. Therefore, we conducted a series of experiments by
varying the amounts of kanamycin added: i) to the fermenter vessel; ii) to the
feed-line and iii) at zero and two hour post-induction. The optimum
kanamycin concentration is 50ug/mL (1X). In order to maintain antibiotic
selection to synthesize the specific protein at higher ODs, either 1X or 2X
kanamycin was used in the vessel. The feed line also contained either 2X or
4X kanamycin, in addition to 0.2X or 0.4X bolus kanamycin at zero and two
hours post induction, with no additional antibiotic added to the feed line or
during induction serving as control.
Figure 3. Comparison of expressed pET19K-
FLYS3 and pET19K-SX-FLYS3. Note that
expression from the SX vector in lane 3 is
substantially greater than that of the 19k vector in
lane 2.
Figure 4. The role of kanamycin for spider silk
protein production is shown here. It can be seen
that there is no drop in plasmid levels when
kanamycin is added at the time of induction (0h).
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10 11 12
OD,600nm
Time, h
Growth curves for FlYS3 protein under
different Kanamycin & IPTG concentrations
1X Kan,(-Kan) FeedLine, 1X IPTG
2X Kan, 2X Kan Feedline, 2X IPTG
1X Kan, 2X Kan Feedline, 2X IPTG
2X Kan, 2X Kan Feedline, 1X IPTG
2X Kan, 4X Kan Feedline, 2X IPTG
1X Kan, 4X Kan Feedline, 2X IPTG
Figure 5. Growth curves for the spider silk
protein FlYS3 at different Kanamycin and
IPTG concentrations are shown here. A higher
OD was achieved when using increased
concentrations of kanamycin and IPTG.
Figure 6. Quantification of bacterially produced
spider silk using ELISA. Samples taken from
fermentation pellet before purification. Protein
concentrations significantly increased from 0.2
g/L to 2.0 g/L after optimization.
• E. coli with pET19K-SX-FLYS3 vector produced more spider silk
protein than E. coli with pET19K-FLYS3.
• Addition of kanamycin study indicates addition of antibiotic is
essential for preventing loss of plasmid at high ODs.
• With the fermentation protocol and our expression vector E.coli can
be grown to high cell density.
• The optimized fermentation protocol increased E.coli growth from
an average OD600 of 30 to 120. Protein concentrations were also
increased f rom 0.2 g/L to 2.0 g/L.
1.8 2.8 0.95 0.81
0
0.5
1
1.5
2
2.5
3
3.5
SX FLYS SX FLYS3 SX FLYS4 SX FLYS6
ELISAquantification,g/LatanOD600100