Overexpression of the Bacillus subtilis and circulans xylanases in Escherichia coli.
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 (Thr147Ser). 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.
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
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Sung
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Luk
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Zahab
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Wakarchuk
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012995:Solubility; D043325:Xylan Endo-1,3-beta-Xylosidase
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase