Sung W L, Luk C K, Zahab D M, Wakarchuk W
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Protein Expr Purif. 1993 Jun;4(3):200-6. doi: 10.1006/prep.1993.1026.
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.
已开发出一种在大肠杆菌中高效表达低分子量木聚糖酶的系统。设计了一个编码成熟环状芽孢杆菌（Bc）木聚糖酶的基因，以模仿大肠杆菌中使用的简并密码子的频率。使用合适的简并密码子来创建多个独特的限制性位点，用于未来的诱变研究。合成基因分两个阶段构建，两个阶段均涉及重叠寡核苷酸的连接。然后通过单个密码子替换（Thr147Ser）将合成的Bc基因转化为枯草芽孢杆菌（Bs）木聚糖酶基因。进一步修饰了包含这两个合成基因的质粒，以便在大肠杆菌中直接表达。在lac启动子的控制下，重组木聚糖酶已在细胞质中以高达300毫克/升的可溶性形式产生。与之前所有涉及天然基因表达的尝试相比，这种效率有了显著提高，在那些情况下木聚糖酶是分泌型的。对我们基因产物的表征表明，纯化的重组产物经过了正确的加工且具有酶活性。
