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

摘要:

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cDNA序列。利用原核表达载体pET-28a构建pET-28a-PDS表达载体;使用PLtac启动子替换T7 启动子构建pET-PLtac-PDS表达载体;合成Mistic序列融合入pET-28a中构建了pET-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cDNA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pET-28a-PDS和pET-PLtac-PDS表达的PDS蛋白表达量低，并以包涵体形式存在;利用pET-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。

Abstract:

Abstract:［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 cDNA sequence of PDS from Dunaliella salina through RACE．First，we utilized prokaryotic expression vector pET-28a to construct pET-28a-PDS vector．Then，we substituted PLtac promoter for T7 promoter in pET-28a to construct pET-PLtac-PDS vector．Last，we constructed pET-Mistic-PDS fusion vector by integrating Mistic sequence into pET-28a． All were transformed into BL21 (DE3) for protein expression.［Results］The 2237-bp full-length cDNA 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 pET-28a-PDS and pET-PLtac-PDS vector，and proteins were mostly expressed in inclusion body．The expression of PDS protein was significantly increased via pET-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.