蛇足石杉赖氨酸脱羧酶基因的克隆、原核表达及其功能分析

Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata

摘要:

赖氨酸脱羧酶 (Lysine decarboxylase, LDC) 是抗老年痴呆药——石杉碱甲生物合成的第一个酶。为了研究蛇足石杉中LDC的特性和功能，以其总RNA为模板，通过RT-PCR扩增得到2个赖氨酸脱羧酶基因LDC1和LDC2，克隆至pMD?19-T中测序发现，两基因同源性为95.3%，分别编码212和202个氨基酸。将两基因引入pET-32a(+)构建重组表达质粒pET-32a(+)/LDC1和pET-32a(+)/LDC2，分别转入BL21(ED3)中进行诱导表达，在30 ℃条件下获得可溶性表达产物Trx-LDC1和Trx-LDC2；采用Ni-NTA亲和层析法纯化目的蛋白，建立酶促反应体系分析其脱羧酶活性，薄层层析 (TLC) 检测表明重组融合蛋白Trx-LDC1和Trx-LDC2均能催化赖氨酸脱羧生成尸胺。利用生物信息学软件分析发现LDC1和LDC2理化性质存在差异，但预测的二级结构和三维结构基本一致。

Abstract:

Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD?19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 ℃, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 ℃ and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.