OVEREXPRESSION OF THE BACILLUS-SUBTILIS AND CIRCULANS-XYLANASES IN ESCHERICHIA-COLI

被引:43
作者
SUNG, WL
LUK, CK
ZAHAB, DM
WAKARCHUK, W
机构
[1] Institute for Biological Sciences, National Research Council of Canada, Ottawa
关键词
D O I
10.1006/prep.1993.1026
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
An efficient expression system for a low-molecular mass xylanase in Escherichia coli has been developed. A gene encoding the mature Bacillus circulans (Bc) xylanase was designed to imitate the frequency of degenerate codons used in E. coli. Appropriate degenerate codons were used to create multiple unique restriction sites for future mutagenesis studies. The synthetic gene was constructed in two stages, both involving ligation of overlapping oligonucleotides. The synthetic Bc gene was then converted to a Bacillus subtilis (Bs) xylanase gene via a single codon substitution (Thrl47Ser). The plasmids containing both synthetic genes were further modified for the direct expression in E. coli. Under the control of the lac promoter, recombinant xylanase has been produced at levels as high as 300 mg/liter in soluble form in the cytoplasm. This efficiency represented a dramatic improvement over all previous attempts involving the expression of the natural genes, with the xylanase being secreted in those cases. Characterization of our gene products indicated that the purified recombinant product was correctly processed and enzymatically active. © 1993 Academic Press. All rights reserved.
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页码:200 / 206
页数:7
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