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Received May 12, 2011
Accepted May 31, 2011
- This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Expression of redesigned mussel silk-like protein in Escherichia coli
1Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea 2Ocean Science and Technology Institute, Pohang University of Science and Technology, Pohang 790-784, Korea
hjcha@postech.ac.kr
Korean Journal of Chemical Engineering, August 2011, 28(8), 1744-1748(5), 10.1007/s11814-011-0140-3
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Abstract
Silks have been used widely for human beings due to their several extraordinary properties. Until now, the studies on silk proteins have mainly focused on spiders and silkworms. Because silk properties are organism-dependent, novel silk protein types can be found and developed through investigation of new silk-bearing organisms. We noticed that marine mussel has silk-like domains containing many repeats with abundance of glycine and alanine. In the present work, we redesigned mussel-derived silk-like gene sequence which contains alternating repeated and nonrepeated regions with optimized codons for Escherichia coli. For successful expression of recombinant mussel silklike protein in E. coli cells, we employed several experimental strategies, including use of strong promoter, cold shock expression, and genetic fusions. We observed significant repression on cell growths by even low expression levels of soluble mussel silk-like proteins in cold shock- and glutathione s-transferase (GST) fusion-based systems. Thus, we finally used baculoviral polyhedrin protein as a fusion partner and successfully expressed insoluble mussel silk-like protein with relatively high expression level and without cell growth repression in E. coli.
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References
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Gosline J, Lillie M, Carrington E, Guerette P, Ortlepp C, Savage K, Phil. Trans. R. Soc. Lond., B357, 121 (2002)
Vendrely C, Scheibel T, Macromol. Biosci., 7, 401 (2007)
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Qin XX, Coyne KJ, Waite JH, J. Biol. Chem., 272, 32623 (1997)
Qin XX, Waite JH, Proc. Natl. Acad. Sci., 95, 10517 (1998)
Coyne KJ, Qin XX, Waite JH, Science, 277(5333), 1830 (1997)
Lombardi SJ, Kaplan DL, J. Arachnol., 18, 297 (1990)
Rising A, Widhe M, Johansson J, Hedhammar M, Cell. Mol. Life Sci., 68, 169 (2011)
Craig CL, Riekel C, Comp. Biochem. Phys., B., 133, 493 (2002)
Guerette PA, Ginzinger DG, Weber BH, Gosline JM, Science, 272(5258), 112 (1996)
Menzella HG, Microb. Cell Fact., 10, 15 (2011)
Winkler S, Kaplan DL, Rev. Mol. Biotechnol., 74, 85 (2000)
Fahnestock SR, Bedzyk LA, Appl. Microbiol. Biotechnol., 47(1), 33 (1997)
Vaillancourt PE, E. coli Gene Expression Protocols., 205, 1 (2003)
Goldstein J, Pollitt NS, Inouye M, Proc. Natl. Acad. Sci., 87, 283 (1990)
Tanabe H, Goldstein J, Yang M, Inouye M, J. Bacteriol., 174, 3867 (1992)
Steczko J, Donoho GA, Dixon JE, Sugimoto T, Axelrod B, Protein Expr. Purif., 2, 221 (1991)
Tamura M, Ito K, Kunihiro S, Yamasaki C, Haragauchi M, Protein Expr. Purif., 78(1), 1 (2011)
Makrides SC, Microbiol. Rev., 60, 512 (1996)
LaVallie ER, McCoy JM, Curr. Opin. Biotechnol., 6, 501 (1995)
Guo WH, Cao L, Jia ZJ, Wu G, Li T, Lu FX, Lu ZX, Protein Expr. Purif., 77(2), 185 (2011)
Harrap KA, Virology., 50, 124 (1972)
Rohrmann GF, J. Gen. Virol., 67, 1499 (1986)
Seo JH, Li L, Yeo JS, Cha HJ, Biotechnol. Bioeng., 84(4), 467 (2003)
Rudolph R, Lilie H, Faseb J., 110, 49 (1996)
Quant RL, Pearson MN, Rohrmann GF, Beaudreau GS, Appl. Environ. Microbiol., 48, 732 (1984)
Wei Q, Kim YS, Seo JH, Jang WS, Lee IH, Cha HJ, Appl. Environ. Microbiol., 71, 5038 (2005)