甲硫氨酸γ-裂解酶基因在大肠杆菌中的高效表达和活性

High expression and activity of an L-methionine γ-lyase gene in Escherichia coli

【背景】为了开发海洋蕴藏的新型微生物资源,本研究团队采用不依赖培养的宏基因组技术,构建了深海宏基因组文库,并对其中的重要基因进行后续研究。【目的】使用来自深海宏基因组文库中的甲硫氨酸γ-裂解酶基因(mgl)在大肠杆菌中高效表达并对其活性进行检测。【方法】将mgl基因克隆到表达载体pET-28a(+)并转化大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,并对表达条件进行优化,获得甲硫氨酸γ-裂解酶(Methionine-lyase,r MGL)的大量表达。亲和层析纯化重组蛋白后对酶的活性进行研究。【结果】亲和纯化后获得大量表达蛋白r MGL,大小与预测的46 kD相符合,并具有很高的裂解L-甲硫氨酸的活性。rMGL能催化L-甲硫氨酸和DL-同型半胱氨酸的裂解,但几乎不作用于L-半胱氨酸和L-胱氨酸,其中对DL-同型半胱氨酸的催化效率比对L-甲硫氨酸的催化效率高,相对活性约为对L-甲硫氨酸催化效率的1.4倍。【结论】来自深海宏基因组文库中的mgl基因能够利用p ET-28a(+)/BL21(DE3)高效表达rMGL。

[Background] In order to develop new microbial resources in the ocean, our laboratory constructed a deep sea metagenomic library by adopting the culture-independent metagenomic technology, and carried out follow-up studies on the important genes. [Objective] To identify and highly express the methionine γ-lyase gene in Escherichia coli from the DNA library of deep-sea sediments. [Methods] The gene mgl was overexpressed by pET-28 a(+) system in E. coli BL21(DE3), which was induced by isopropyl β-D-1-thiogalactopyranoside, and the expression conditions were optimized to obtain the high expression of methionine-lyase(rMGL). The recombinant protein was purified by affinity chromatography and the enzyme activity was studied. [Results] The product rMGL was consistent with the predicted 46 k D, with high L-methionine lyase activity. rMGL could use L-methionine and DL-homocysteine as substrate, but had little activity on L-cysteine and L-cystine. Its relative activity on DL-homocysteine was 1.4 times of that on L-methionine. [Conclusion] mgl gene from the deep sea metagenomic library can efficiently express rMGL using pET-28 a(+)/BL21(DE3).