人眼晶状体醛脱氢酶1的克隆表达和酶特性研究

目的:研究人眼ALDH1A1的结构与功能学特点。方法:使用标准的PCR技术,从人晶状体cDNA文库扩增ALDH1A1。扩增的ALDH1A1(1.5kb)被插入到克隆表达载体pET-28b,再转染到菌株BL21DE3中。表达出来的蛋白质经His-tag柱纯化,使用thrombin切除His-tag。对纯化的ALDH1A1的酶学特点,如酶活性与辅酶、缓冲液、还原剂、pH以及析出方式等的关系进行了检测。结果:从人晶状体的基因文库,成功扩增了ALDH1A1,重组的人晶状体ALDH1A1为1506bp的DNA及501个氨基酸组成,分子量为54.8kDa,PI=6.8。与人的肝脏AL-DH1A1100%同源,与大鼠和小鼠肝脏的ALDH1A1有85%的同源性。Thrombin酶切较imidazole洗脱的ALDH1A1活性保持得更持久,纯化的ALDH1A1在碱性的sodiumpyrophosphate反应缓冲液中,NAD为辅酶和dTT作为还原剂时的活性较高,其活性随pH增高而增加。结论:ALDH1A1与人肝组织的同工酶有相似的作用。

Molecular cloning expression and biochemical characterization of the human lens ALDH1A1

AIM:To clone, express and characterize the biochemi-cal properties of recombinant human lens ALDH1A1 protein. ·METHODS: The complete coding sequence of ALDH1A1 from human lens library was cloned and expressed in E. coli. with PCR and plasmid transformation. Recombinant human lens ALDH1A1 protein was purified using His-tag column, and its biochemical properties such as enzyme activity, reaction buffer, cofactor, reductant and pH were studied. ·RESULTS: The coding region of human lens ALDH1A1 cDNA encodes a 1506bp DNA and a 501 amino acid protein (MW=54.8 kDa) that is 100% identical to human liver ALDH1A1 and shares a 85% with rat and mouse liver. The activity of recombinant human lens ALDH1A1 can be maintained for a longer period while eluted by thrombin than by imidazole, which has optimum activity with sodium pyrophosphate as a reaction buffer at pH 8.0, preferring NAD as cofactor and dTT as reductant, and exhibits increase activity with higher pH.·CONCLUSION: ALDH1A1 exhibits similar characters as human liver ALDH1A1.