Journal
PROCESS BIOCHEMISTRY
Volume 51, Issue 4, Pages 484-490Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2016.01.006
Keywords
Spider dragline silk; Escherichia coli; Recombinant protein production; Repetitive protein; High cell density cultivation; Synthetic biology approach
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Funding
- National Natural Science Foundation of China [31470216, 21176153]
- Shanghai Pujiang Program [14PJ1405200]
- National Basic Research Program of China (973 Program) [2012CB721006]
- Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning [ZXDF080005]
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Spider dragline silk exhibits excellent mechanical properties that make it a promising protein polymer for industrial and biomedical applications. Since farming spiders is not feasible due to their highly territorial nature, recombinant production of dragline silk proteins in a foreign host has received great attention. However, their production titer remains low, because efficient expression of very large, highly repetitive, glycine-rich silk proteins is a challenge. This work demonstrates the design and high-level production of large dragline silk proteins of major ampullate spidroin 2 (MaSp2) in Escherichia coli by synthetic biology approach. The expression levels of MaSp2 with molecular weight of 28.3-256.5 kDa were significantly elevated by down-shifting the induction temperature. The beneficial effect was found to be at least partially attributed to the improved plasmid maintenance in the recombinant cells. Combination of induction temperature downshift with the glycyl-tRNA pool increase in E. coli led to enhanced biosynthesis of glycine-rich silk proteins. A high production titer of about 3.6 gl(-1) of a 201.6-kDa MaSp2 protein was achieved in a 3-L fed-batch bioreactor, which was the highest as reported. The developed approach may be useful to cost-effective large-scale production of silk proteins. (C) 2016 Elsevier Ltd. All rights reserved.
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