One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.
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Research Article|
February 26 2007
Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli
Shimin Jiang;
Shimin Jiang
*Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
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Chunhong Li;
Chunhong Li
*Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
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Weiwen Zhang;
Weiwen Zhang
†Microbiology Department, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, U.S.A.
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Yuanheng Cai;
Yuanheng Cai
*Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
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Yunliu Yang;
Yunliu Yang
*Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
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Sheng Yang;
Sheng Yang
*Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
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Weihong Jiang
Weihong Jiang
1
*Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China
‡Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, People's Republic of China
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
September 25 2006
Revision Received:
November 20 2006
Accepted:
November 23 2006
Accepted Manuscript online:
November 23 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2007
Biochem J (2007) 402 (3): 429–437.
Article history
Received:
September 25 2006
Revision Received:
November 20 2006
Accepted:
November 23 2006
Accepted Manuscript online:
November 23 2006
Citation
Shimin Jiang, Chunhong Li, Weiwen Zhang, Yuanheng Cai, Yunliu Yang, Sheng Yang, Weihong Jiang; Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli. Biochem J 15 March 2007; 402 (3): 429–437. doi: https://doi.org/10.1042/BJ20061457
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