家蚕吡哆醛激酶的融合表达与纯化

Fusion expression and purification of pyridoxal kinase of Bombyx mori

【目的】吡哆醛激酶（pyridoxal kinase,PLK,EC2.7.1.35）是维生素B6的关键代谢酶。本研究原核表达家蚕Bombyxmori重组PLK,为进一步开展家蚕PLK的催化作用机制和表达调控机制的研究奠定基础。【方法】构建家蚕PLK基因融合表达质粒,转化大肠杆菌Escherichia coli诱导表达,经Ni2＋亲和层析纯化后,对融合蛋白的催化活性进行分析。【结果】纯化后的家蚕重组PLK经SDS-PAGE鉴定为单一条带,比活力为1800U/mg,纯化倍数为40倍。在底物过量的条件下,该重组酶的体外最适反应温度是50℃;最适pH为5.5~6;Zn2＋是酶促反应有效的激活剂。【结论】重组家蚕PLK与来源于家蚕组织的PLK具有相同的催化性质。

[Aim] Pyridoxal kinase（PLK,EC 2.7.1.35）is a key enzyme related to vitamin B6 metabolism.In this study,recombinant pyridoxal kinase of Bombyx mori will be expressed,which will lay a foundation for further study on regulation mechanism,catalytic mechanism and structure of PLK in B.mori.[Methods] After fusion expression vector harboring PLK gene of B.mori was constructed,the recombinant vector was transformed to Escherichia coli Rosetta for induction and expression,and then the characteristics of the purified recombinant protein was analyzed after the expression product was purified using affinity chromatography by Ni2＋ column.[Results] Only one band was observed when the purified PLK was separated on SDS-PAGE.The PLK was enriched by 40-fold and its specific activity was 1 800 U/mg.The optimum temperature and pH of this enzyme was about 50℃,and 5.5-6,respectively,and Zn2＋ was the effective activator in enzymatic reaction.[Conclusion] The recombinant PLK exhibited the same catalytic characteristics as the native protein obtained from tissues of B.mori.