Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide.
Appl Microbiol Biotechnol, 2010/2;85(5):1473-81.
Fang N[1], Zhong CQ, Liang X, Tang XF, Tang B
Affiliations
PMID: 19697018DOI: 10.1007/s00253-009-2183-5
Impact factor: 5.56
Abstract
Limited secretion capacity remains a drawback of using Escherichia coli as the host for the production of recombinant proteins. In this report, random mutagenesis was performed within the N-terminal propeptide of thermostable WF146 protease, a subtilase from thermophilic Bacillus sp. WF146, generating a variant named WBM(MT) with improved capacity for extracellular production when expressed in E. coli. Two mutations, L(-57)Q and E(-10)D, were identified within the N-terminal propeptide. The amount of WBM(MT) in the culture medium was found to be about three times higher than that of wild type. Besides, the introduction of mutations L(-57)Q/E(-10)D into the N-terminal propeptide also accelerated the maturation of the enzyme. Biochemical analysis indicated that the thermostability and the catalytic activity of mature WBM(MT) were similar to those of wild type. Far-UV CD spectra analysis and limited proteolysis experiments suggested that the mutations L(-57)Q/E(-10)D resulted in a structural change in the N-terminal propeptide of the proform, and the N-terminal propeptide became more flexible, which might be beneficial for the proform to keep in a translocation-competent state. Our result indicates that N-terminal propeptide engineering may be a valuable approach for improving extracellular production of recombinant subtilases expressed in E. coli.
MeSH terms
Amino Acid Sequence; Bacillus; Bacterial Proteins; DNA, Bacterial; Enzyme Stability; Escherichia coli; Extracellular Space; Hot Temperature; Molecular Sequence Data; Mutagenesis; Mutation; Protein Engineering; Protein Folding; Protein Precursors; Protein Processing, Post-Translational; Recombinant Proteins; Sequence Alignment; Serine Endopeptidases
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