盐藻PDS基因的同源克隆及在大肠杆菌中的表达  

Homologous cloning and expression of PDS gene from Dunaliella salina in Escherichia coli

作　　者：周峰[1] 黄非[1] 白林含[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,四川成都610064

出　　处：《微生物学报》2015年第2期149-155,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30970043)~~

摘　　要：【目的】八氢番茄红素脱氢酶PDS为真核膜结合蛋白,我们通过更换不同的表达策略,探索在大肠杆菌中表达真核膜结合蛋白的方式。【方法】利用RACE的方法克隆盐藻PDS的全长c DNA序列。利用原核表达载体p ET-28a构建p ET-28a-PDS表达载体;使用PLtac启动子替换T7启动子构建p ET-PLtacPDS表达载体;合成Mistic序列融合入p ET-28 a中构建了p ET-Mistic-PDS融合表达载体。分别转化入大肠杆菌BL21(DE3)中进行原核表达。【结果】获得了盐藻PDS基因的全长c DNA序列2237 bp,开放阅读框为1749 bp,共编码582个氨基酸(NCBI登录号为GQ923693.1)。利用p ET-28a-PDS和p ET-PLtacPDS表达的PDS蛋白表达量低,并以包涵体形式存在;利用p ET-Mistic-PDS载体表达的PDS蛋白表达量明显提高,且大部分以可溶蛋白形式存在,具有脱氢酶活性。【结论】实验结果表明Mistic作为促溶标签能促进膜蛋白的正确折叠,提高蛋白的可溶性。蛋白酶活测定结果证明了Mistic的融合可以保持蛋白的天然活性。[Objective] Phytoene desaturase PDS is a eukaryotic nuclear membrane binding protein,we used different expression methods to search for the soluble expression strategy of membrane binding protein in Escherichia coli.[Methods]We cloned the full-length c DNA sequence of PDS from Dunaliella salina through RACE. First,we utilized prokaryotic expression vector p ET-28 a to construct p ET-28a-PDS vector. Then,we substituted PLtac promoter for T7 promoter in p ET-28 a to construct p ET-PLtac-PDS vector. Last, we constructed p ET-Mistic-PDS fusion vector by integrating Mistic sequence into p ET-28 a. All were transformed into BL21（ DE3） for protein expression. [Results] The2237-bp full-length c DNA sequence of PDS was cloned,including a 1749-bp open reading frame,encoding 582 amino acids（ NCBI accession： GQ923693. 1）. The expression of PDS protein was low via p ET-28a-PDS and p ET-PLtac-PDS vector,and proteins were mostly expressed in inclusion body. The expression of PDS protein was significantly increased via p ET-Mistic-PDS vector,in addition most were expressed as soluble protein which possessed dehydrogenase activity.[Conclusion] Mistic as the solubilization label was able to promote proper folding of membrane proteins and improve solubility. Protease activity assay proved that Mistic could maintain the enzyme activity.

关 键 词：盐藻PDS基因 同源克隆 原核表达 PLtac启动子 Mistic序列 

分 类 号：Q943.2[生物学—植物学]