This document discusses the large-scale production and purification of synthetic spider silk proteins using E. coli. Researchers cloned and expressed spider silk genes from the golden orb-weaving spider Nephila clavipes in E. coli. They tested different genetic vectors (SX and 19K) and protein constructs (MaSP1, FlAS3, FlYS3) and found that the SX vector produced more consistent growth and higher optical densities during fermentation. SDS-PAGE and western blot analysis confirmed production of the target proteins. Optical density curves showed the 19K vector grew faster with the FlYS3 construct. The goal is to quantify and test the mechanical properties of the synthesized spider silk proteins for commercial applications.

1. Spider silk is considered to be the toughest
biomaterial, whose mechanical strength far exceeds
that of steel and Kevlar, and finds attractive
commercial applications ranging from specialty
ropes to medical materials. Owing to the difficulties
in its production using spiders, alternative host
systems and engineering methods have been
investigated to develop suitable production systems
that can efficiently produce spider silk protein.
Escherichia coli is the most widely investigated
heterologous host system due to its extensive use in
other genetic recombination schemes, allowing
straightforward gene manipulation and production
through well-known fermentative processes. Several
bioengineered proteins inspired by the golden orb-
weaving spider Nephila clavipes, have been cloned,
expressed and purified successfully. Here, we show
that proteins of different molecular weights ranging
from 30–90 kDa have been fermented at 10L scales
with optical densities reaching 80–120 and purified
using affinity chromatography. Upon production of
sufficient quantities of synthetic spider silk, we will
next explore the structure-function properties of
these biomaterials for functional outcomes.
Large scale production and purification of
chimeric spider silks in Escherichia coli
Jordan M. Wanlass, R. Chase Spencer, Sreevidhya T. Krishnaji, Paula F. Olivera, Justin A. Jones, Randolph V. Lewis Utah State University
The large scale optimization of synthetic silk
production using E. coli requires a genetic vector
and protein construct that will produce in large
quantities. By measuring the Optical Density (OD)
during fermentation, decisions can be made as to
which vector provides better growth. Two such
vectors used in this process are named as SX and
19K. Both will be tested with three essential protein
constructs: the FlAS3 and FlYS3 are the constructs
responsible for the elasticity of the silk, while MaSP1
contributes to its strength. The methods in Figure 2
were implemented using combinations of these
vectors and constructs (see Figure 1).
The graphs display the absorbance taken with a
spectrophotometer reading at 600 nanometers (see
Figure 4). A higher growth rate and an overall more
consistent growth was achieved from the 19K vector
within the FlYS3 construct, but the SX vector is more
consistent in giving us desired results of high optical
densities per unit time.
The SDS-PAGE (Sodium Dodecyl Sulfate
Polyacrylamide gel electrophoresis) and the
Western blots (protein immunoblot) confirm the
existence of proteins at the desired sizes for MaSP1
and FlAS3 (see Figure 3). This indicates that our
modified E. Coli cells are producing protein through
those highlighted constructs and vectors (see Figure
1).
SX vector gives a more consistent OD curve and
generally higher values than 19K. This could be due
to the fact that the components for making Gly and
Pro tRNA’s are present in SX and not in 19K. Our
next step will be to quantify the lyophilized spider
silk proteins and fibers will undergo various material
tests and be passed into a diverse range of
applications.
R. Chase Spencer
Utah State University
Biological Engineering
robertchasespencer@gmail.com
I. Introduction II. Methods III. Results
IV. Conclusions
High speed
centrifugation by
continuous flow
Bacterial mass (pellet)
Affinity chromatography of
10X Histidine tagged
proteins by Äkta
Confirm protein presence
through SDS/Western blot
Jordan Wanlass
Utah State University
Biological Engineering
Jordan.wanlass@gmail.com
Funding: USTAR (Utah Science Technology and
Research), NSF (National Science Foundation), and DOE
(Department of Energy).
Special Thanks to Matthew C. Sims, Christopher Peterson
and Dong Chen for assistance and advice.
7h 8h 9h 10h 11hM
M 6h 6.5h 8h 9h 10h 11h 12h
Constructs Mol. Wt. [kDa]
Flag-like A4S88 70
FlAS 2X 51
3X 74
4X 97
FlYS 2X 57
3X 83
4X 109
MaSp1 16X 70
24X 100
32X 150
0.000
20.000
40.000
60.000
80.000
100.000
120.000
140.000
160.000
0.0 2.0 4.0 6.0 8.0 10.0 12.0
OpticalDensity,600nm
Time (hrs.)
Optical Density Curves of 19K Vector
19K MaSp1
19K MaSp1
19K FlAS3
19K FlAS3
19K FlSY3
19K FlSY3
0.000
10.000
20.000
30.000
40.000
50.000
60.000
70.000
80.000
90.000
100.000
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
OpticalDensity,600nm
Time (hrs.)
Optical Density Curves of SX Vector
SX MaSp1
SX MaSp1
SX FlAS3
SX FlAS3
SX FlSY3
SX FlSY3
Fermentation on a 5, 10 and 100 L scale
Lyophilization for final
protein product
Cloning and starter culture preparation
with highlighted constructs
V. Acknowledgements
Figure 1:
Figure 2:Figure 3:
Figure 4:
Construct Sequence
MaSP1 (1x) GAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAA
FlAS (1x) GPGGAGPGGA GPGGAGPGGA GPGGAGPGGA GPGGAGPGGA
GPSGPGSAAA AAAAA
FlYS (1x) GPGGPGGYGP GGSGPGGYGP GGSGPGGYGP GGSGPGGYGP
GGSGPSGPGS AAAAAAAA