title: Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli.
abstract: 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 beta-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.
authors.affiliation: 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.
authors.forename: Shimin
authors.initials: S
authors.lastname: Jiang
authors.forename: Chunhong
authors.initials: C
authors.lastname: Li
authors.forename: Weiwen
authors.initials: W
authors.lastname: Zhang
authors.forename: Yuanheng
authors.initials: Y
authors.lastname: Cai
authors.forename: Yunliu
authors.initials: Y
authors.lastname: Yang
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D019143:Evolution, Molecular; D015870:Gene Expression; D014644:Genetic Variation / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D040681:Structural Homology, Protein; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D007527:Isoenzymes; D011994:Recombinant Proteins; D000581:Amidohydrolases; C085345:1-carbamoyl-L-amino acid amidohydrolase