极端耐热木聚糖酶基因在大肠杆菌中的高效表达

Expression of Xylanase B Gene of Thermotoga maritima in Escherichia coli

海栖热袍菌Thermotogamaritima为嗜极端高温的厌氧细菌 ,其产生的极耐热性木聚糖酶B具有非常可观的工业应用前景。但编码该酶的基因xynB在大肠杆菌中的表达较困难。文中利用PCR技术将xynB克隆至原核表达载体pET 2 0b ,并与组氨酸标签进行融合构建成重组质粒 pET 2 0 xynB。研究发现 ,pET 2 0 xynB在稀有密码子AGA ,AGG和AUA得到加强的大肠杆菌宿主菌株BL2 1 CodonPlus(DE3 ) RIL中 ,木聚糖酶基因的表达水平远远高于在其他菌株中的表达。结合对重组菌的诱导条件的优化 ,使得重组蛋白表达量达到11 5 % ,比酶活达到 2 5 69U/mg ,比常规基因工程菌株提高 12 5倍。

Thermotoga maritima is one of the few hyperthermophilic bacteria described to date. The xylanases found in T. maritima showed extremely high thermostability and considerable potential in industrial application. The gene of xylanase B from T. maritime ATCC 43589 was amplified by PCR, inserted into the plasmid pET-20b, and fused with the 6-His tag. The recombinant plasmids pET-20b-xynB were transformed to E. coli BL21-CodonPlus(DE3)-RIL. Rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of xynB gene in E.coli. It was found that the activities of recombinant xylanase was 12.5 times higher in BL21-CodonPlus(DE3)-RIL than that in conventional E.coli host strains under optimized induction conditions.