拟南芥Antiquitin基因的原核表达和生物信息学分析  

Prokaryotic Expression and Bioinformatics Analysis of Antiquitin Protein from Arabidopsis thaliana

作　　者：安瑞[1] 魏少华[1] 范三红[1,2] 单丽伟[2] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨陵712100 [2]陕西省农业分子生物学重点实验室,陕西杨陵712100

出　　处：《西北植物学报》2009年第12期2378-2384,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(30300222);西北农林科技大学优秀人才基金(042R009)

摘　　要：将拟南芥Antiquitin基因重组到原核表达载体pMAL-c4x和pET41中,在T7 Express菌株中诱导表达,经Amylose和Ni-NTA亲和层析柱纯化获得重组蛋白。SDS-PAGE结果表明:MBP和His-tag融合的拟南芥An-tiquitin主要以可溶性形式存在,表达量分别占细胞总蛋白的25.1%和39.4%。以乙醛和NAD+为底物测定融合蛋白活性,结果显示:His-tag融合的Antiquitin具有醛脱氢酶活性,比活力为8.98 U/mg,乙醛的表观Km和Vmax值分别为0.98 mmol/L和12.75 U/mg。序列比对和结构预测结果显示:拟南芥Antiquitin包含该家族蛋白典型的催化结构域、NADH结合结构域和寡聚化结构域,活性中心残基为Gly238、Gly291、Glu391、Phe393。The encoding sequence of Antiquitin protein from Arabidopsis thaliana was cloned into pMAL-c4x and pET41 vector.Fusion proteins were expressed in E.coli strain T7 Express and purified by amylose resin column and Ni-NTA resin column.Based on the results of SDS-PAGE,the yield of fusion protein,which tagged by MBP and His6,accounts for 25.1% and 39.4% of the total bacterial protein,respectively.The aldehyde dehydrogenase activity of the purified protein was determined using acetaldehyde and NAD＋ substrates.The result showed that the soluble fusion protein from Ni-NTA resin column had detectable aldehyde dehydrogenase activity and the specific activity is 8.98 U/mg.The Km and Vmax for acetaldehyde are 0.98 mmol/L and 12.75 U/mg,respectively.Sequence alignment and structure prediction reveals：Arabidopsis Antiquitin may exist as an oligomer,each monomer is comprised of three domains,catalytic domain,NAD＋-binding domain and oligomerization domain,and the active residues are Gly238,Gly291,Glu391 and Phe393.

关 键 词：拟南芥 ANTIQUITIN 重组表达 亲和纯化 NADH 

分 类 号：Q811.4[生物学—生物工程]