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Recovery of N-carbamoyl-L-amino acid amidohydrolase from inclusion bodies
|關鍵字:||N-胺甲醯基-L型-胺基酸胺基水解酵素;refolding;穀胱甘肽;N-carbamoyl-L-amino acid amidohydrolase;GSH;GSSG||出版社:||化學工程學系所||引用:|| S. C. Lu and S. C. Lin, "Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers," Enzyme Microb Technol, vol. 50, pp. 65-70, 2012.  M. M. Carrio and A. Villaverde, "Construction and deconstruction of bacterial inclusion bodies," J Biotechnol, vol. 96, pp. 3-12, 2002.  M. Merdanovic, T. Clausen, M. Kaiser, R. Huber, and M. Ehrmann, "Protein quality control in the bacterial periplasm," Annu Rev Microbiol, vol. 65, pp. 149-68, 2011.  A. Mitraki, B. Fane, C. Haase-Pettingell, J. Sturtevant, and J. King, "Global suppression of protein folding defects and inclusion body formation," Science, vol. 253, pp. 54-8, 1991.  M. Li, Z. G. Su, and J. C. Janson, "In vitro protein refolding by chromatographic procedures," Protein Expr Purif, vol. 33, pp. 1-10, 2004.  S. C. Lin, K. L. Lin, H. C. Chiu, and S. 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利用大腸桿菌生產N-胺甲醯基-L型-胺基酸胺基水解酵素時，會因大量表現而產生內函體。利用Tris buffer直接溶解內函體後即可得到活性蛋白。其中以pH 9.0所回收得來的蛋白比活性0.40 U/mg為最高，但還是不如native蛋白(pH 8.0下比活性為1.58 U/mg，pH 9.0下為1.31 U/mg)。將native蛋白加入DTT後可發現比活性迅速降低，顯示雙硫鍵對於比活性具有重大影響。而從內函體中回收的可溶蛋白不具有雙硫鍵結構，所以缺乏正確的雙硫鍵結構可能是導致活性不如native蛋白的原因。
由於雙硫鍵對於蛋白活性具有重大影響，故在後續進行復性實驗時加入GSH/GSSH以幫助雙硫鍵的形成。在復性緩衝溶液中添加總濃度[GSH]+2[GSSG]=10 mM，比例[GSH]/2[GSSG]=4的情況下，可將可溶蛋白回收率從80.0%提升10.3%至90.3%。添加總濃度[GSH]+2[GSSG]=10 mM，比例[GSH]/2[GSSG]=10或8的情況下，則可將總活性從0.021 U提升76%至0.037 U。
Recombinant N-carbamoyl-L-amino acid amidohydrolase was over-expressed in Escherichia coli in the form of inclusion bodies. Active protein could be recovered from inclusion bodies by direct solubilization with Tris buffer. The largest specific activity of solubilized protein is 0.40 U/mg at pH 9.0, but it was still lower than native protein, 1.58 U/mg at pH 8.0 and 1.31 U/mg at pH 9.0. The result of adding DTT in native protein indicated that disulfide bond structures had significant impact on specific activity. The structures of solubilized proteins from inclusion bodies didn’t have disulfide bond structures, so lack of right disulfide bond structures may be the reason of lower specific activity.
Because of the significance of disulfide bond structures, GSH/GSSH was added into the refolding system to assist formation of disulfide bonds. Recovery of soluble protein could be increased by 10.3% from 80.0% to 90.3% when the refolding buffer contained 10 mM of a 4:1 ratio of [GSH]/2[GSSG]. Total activity could be increased by 76% from 0.021 U to 0.037 U when the refolding buffer contained 10 mM of a 10:1 or 8:1 ratio of [GSH]/2[GSSG].
Overall, adding GSH/GSSG could enhance refolding result of N-carbamoyl-L-amino acid amidohydrolase.
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