蚓激酶基因F238在大肠杆菌中的可溶性表达

Soluble expression of lumbrokinase F238 gene in E.coli.

应用PCR技术扩增获得蚓激酶成熟肽基因F238,并将其克隆至大肠杆菌质粒pFT22bTrxA中,构建双顺反子表达载体pTrx-F238。重组质粒转化大肠杆菌BL21(DE3),1.0mmol/LIPTG、30℃条件下诱导6h,经SDS-PAGE证实蚓激酶成熟肽蛋白获得了高效表达,所表达的蛋白产物相对分子量与预期的26kDa相符,且都是可溶性蛋白,表明分子伴侣TrxA起到了帮助目的蛋白正确折叠的作用。利用血纤维蛋白法对产物活性进行了测定,确定其不仅具有激酶作用,而且具有直接溶栓活性。最高活力可达180U/mL(尿激酶单位)。

The mature peptide gene F238 of lumbroldnase was amplified by PCR and cloned into vector pET22b-Trx to construct expression plasmid pTrx-F238 which was transformed into E. coli BL21（DEB）. The recombinant was inducted for 6 h under the condition of 1.0 mmol/L WIG at 30 ℃ PAGE suggested that the mature peptide protein of lumbrokinase was expressed in high level and the molecular weight of the target protein was about 26 kDa, in correspondence with the theoretical value. The lumbrokinase was successfully expressed in the form of soluble protein indicated that TrxA played a key role in the folding process of the target protein. The fibrinolytic activity of the lumbrokinase expressed in E. coli was measured using artificial fibrin plates. The result indicated that lumbrokinase could directly dissolve fibrous protein, and also had the activity of fibrinokinase which could indirectly dissolve fibrous protein. The activity of lumbroldnase was up to 180 U/mL.