large scale production and purification of chimeric spider
TRANSCRIPT
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 MaSP1contributes 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 MaSP1and 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 SpencerUtah State UniversityBiological [email protected]
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 WanlassUtah State UniversityBiological [email protected]
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 70FlAS 2X 51
3X 744X 97
FlYS 2X 573X 834X 109
MaSp1 16X 7024X 10032X 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
Opt
ical
Den
sity
, 600
nm
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
Opt
ical
Den
sity
, 600
nm
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