重组蛋氨酸裂解酶原核体系的表达及纯化

The Expression and Purification of Recombinant Methionine Lyase in the Prokaryotic System

目的建立L-蛋氨酸γ-裂解酶的原核表达、纯化体系.方法合成Metase基因并构建重组质粒pBSK-Metase.通过分子克隆技术,构建重组表达质粒pBV220-Metase和pGEX-4T-1-Metase,并将重组子转化到感受态大肠杆菌Dh5α中.pGEX-4T-1-Metase经异丙基-β-D-硫代半乳糖苷(IPTG)诱导;pBV220-Metase经42℃温度诱导;并对诱导条件进行优化,获得大量表达;建立蛋氨酸裂解酶纯化体系.结果构建出Metase基因的两种高效原核表达体系pBV220-Metase和pGEX-4T-1-Metase,并优化各自的最佳诱导表达条件;建立重组蛋氨酸裂解酶的纯化体系.结论成功建立重组蛋氨酸裂解酶的原核表达、纯化体系;获得高效原核表达重组子pGEX-4T-1-Metase,且获得纯度高达95%的蛋氨酸酶.

Objective To construct a prokaryotic expression and purification system of the L-methionine y -lyase. Methods Metase gene was synthesized and a gene recombinant plasmid pBSK-Metase was constructed. The expression plasmids pBV220-Metase and pGEX-4T-1-Metase were constructed by molecular cloning technology, and were transformed into e. coli Dh5 or. The expression of pGEX-4T-1-Metase was induced by IPTG and pBV220-Metase was induced by incubation at 42~C, the induction conditions were optimized. Through the study of the target protein purification, the methionine repressible enzyme purification system was established. Results Two highly efficient prokaryotic expression and purification system pBV220-Metase and pGEX-4T-1-Metase were established and their induction conditions were optimized. The methionine lyase purification system was established. Conclusion A prokaryotic expression and purification system of the L-methionine Y-lyase is established successfully, and pGEX-4T-1-Metase could express well and get purity as much as 95% of methionine enzymes.