利用pET-29b载体进行葡激酶基因表达与其产物的亲和纯化

目的：探讨6聚组氨酸序列与葡激酶基因序列融合表达及其表达产物的纯化方法。方法：将葡激酶的cDNA序列连入pET-29b质粒，转入E.coli BL21（DE3）进行表达。应用镍金属离子螯合层析及凝胶过滤层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定。结果：6聚组氨酸与葡激酶的可溶性融合蛋白在BL21（DE3）中的表达量约占菌体可溶性蛋白总量的25％；螯合层析纯化后其纯度可达90％以上；再应用Sephacryl S-200HR可使其纯度提高到98％以上；测得其比活性为5.0×10⁴ AU•mg^-1。结论：利用pET-29b载体成功进行了重组葡激酶基因的表达及其表达产物的亲和纯化。

Expression and affinity purification of recombinant staphylokinase using pET-29b vector

Objective To investigate the fusion expression of staphylokinase(SAK) gene with (His) 6-tag and the purification of the expression product with Ni 2+ - metal chelating affinity chromatography. Methods The fragment of SAK cDNA was inserted into the pET-29b expression plasmid and the recombinant vector pET-29b-SAK was transformed into E. coli BL21(DE3) which was then induced by IPTG. The recombinant SAK-(His) 6 was purified with His·Tag affinity chromatography, and its bioactivity was analyzed. Results SDS-PAGE analysis indicated that the soluble fusion protein occupied 25% of the total soluble proteins in BL21(DE3). By one-step metal chelating chromatography using Ni-IDA resin, the purity of rSAK-(His) 6 was more than 90%. Then the recombinant target product obtained greater than 98% purity by the sequent exclusion chromatography using Sephacryl S-200HR. The specific activity of rSAK-(His) 6 purified was more than 5.0×10 4 AU·mg -1 . Conclusion rSAK-(His) 6 has been expressed and purified successfully using pET-29b vector.