人ppGalNAc-T2的原核表达、纯化及其蓖麻蛋白样结构域的结构预测

多肽∶N乙酰氨基半乳糖转移酶(ppGalNAcT)是催化O糖基化的起始酶,在O聚糖的形成中起着关键的作用.为更好地研究该家族酶的结构与功能,采用PCR技术从pDONR201T2得到ppGalNAcT2全长编码序列,亚克隆至原核表达载体pGEX4T1,转化大肠杆菌BL21,获得相应表达产物,用谷胱甘肽S转移酶(GST)亲和层析柱进行纯化,并进行了Western印迹检测和初步的酶活测定.为进一步研究其功能还在结构研究上利用网络结构模拟SWISSMODEL服务器对ppGalNAcT2的蓖麻蛋白样结构域进行结构模拟.成功构建了原核表达载体pGEX4T1T2,获得有效表达和纯化,并初步鉴定了其活性,同时预测了其可能的三维结构和活性位点.

Prokaryotic Expression,Purification of Human ppGalNAc-T2 and Structure Simulation for the Ricin-like Domain

Polypeptide∶N-acetylgalactosaminyl transferase (ppGalNAc-T) is the primary enzyme of O-glycosylation and plays an important role in forming O-polysaccharide. To investigate the functional and structure of the enzyme familiy further,the target DNA fragment enconding ppGalNAc-T2 was amplified from cloning plasmid pDONR201-T2 by PCR and sub-cloned into the prokaryotic expression vector pGEX-4T-1,the recombinant vector was introduced into E.coli BL21 for efficient expression. The GST-fusion protein was purified through GST-column. Analysis for the recombinant protein with Western blot showed a single band as expected.Using deduced ppGalNAc-T2 amino acid sequences in TBLAST search of human genomic DNA database identified several sequences,among them the highest homology was ricin-like domain,alignments between ppGalNAc-T2 and ricin-like domain were optimal in Swiss-PDB Viewer,finally examination with PROCHEK revealed that the molecular modeling of the enzyme has 91.4% of amino acid residues located in optimal region providing the O-glycosylation location.