枯草杆菌谷氨酰胺转胺酶的克隆及其在大肠杆菌中的融合表达

Cloning of the Transglutaminase Gene from Bacillus subtilis and Fusion Expression in Escherchia coli

利用PCR技术 ,从枯草杆菌DB40 3染色体上扩增出谷氨酰胺转胺酶基因 ,将其克隆到大肠杆菌载体pET32a( + )中 ,成功构建谷氨酰胺转胺酶表达载体pET32 BTGase ,并转化大肠杆菌BL2 1 (DE3)。重组克隆在IPTG诱导下 ,表达出硫氧还蛋白 谷氨酰胺转胺酶 (Trx BTGase)融合蛋白 ,表达量占细菌总蛋白量的 2 6%。利用金属螯合层析纯化菌体裂解上清中表达的融合蛋白 ,纯度超过 80 %,再通过分子筛层析进一步纯化得到融合蛋白纯品。酶活性分析表明表达的Trx BTGase融合蛋白具有交联蛋白的活性 ,并发现Trx

The gene of transglutaminase was amplified from the chromosome of Bacillus subtilis DB403 by PCR and translationally fused with thioredoxin gene ( trx A) in the plasmid pET32a(+) to achieve the expression vector pET32 BTGase. The fusion protein Trx BTGase could be expressed in E. coli BL21 (DE3) after IPTG induction, with a level up to 26 percent of the total bacterial proteins. Trx BTGase was initially purified from the supernatant of the bacterial lysate by metal chelating chromatography with a purity of over 80 percent, and finally purified with Superdex 75. Enzyme activity assay demonstrated that the Trx BTGase expressed in E. coli could be functional in polymerizing other proteins, and no influence on the biological activities of BTGase to polymerize BSA (bovine serum albumin) was found in case of the thrombin cleavage of this fusion protein.;