低分子量麦谷蛋白基因XYGluD3-LMWGS1原核表达增溶体系的探索

为研究SUMO标签增溶体系和小麦低分子量麦谷蛋白亚基功能,利用SUMO标签构建XYGluD3-LM-WGS基因原核表达可溶性增强的系统,采用PCR方法从已有质粒XYGluD3-LMWGS1/pGEM-Teasy中克隆到该基因不含上游启动子片段XYGluD3-LMWGS1-no Promoter 798 bp(简称LnoP),构建原核表达载体LnoP/pGEX-4T-1,并借助已有质粒SUMO1/pET-41a( )构建原核表达载体LnoP-SUMO1/pET-41a( )与LnoP-SUMO1-N2/pET-41a( )以及LnoP-SUMO1/pET-32a( ),分别诱导表达融合蛋白分析其可溶性,结果显示;(1)融合蛋白GST-LnoP、GST-SUMO1-LnoP、SUMO1-LnoP和Trx-SUMO1-LnoP表达成功;(2)电泳和Western blot分析显示,GST-LnoP和GST-SUMO1-LnoP的可溶蛋白表达均为电泳不可见,SUMO1-LnoP与Trx-SUMO1-LnoP有微量可溶表达.结果表明,试验首次实现人的SUMO1标签在麦谷蛋白低分子量亚基中的融合表达;尽管人的SUMO1做为融合标签对于增加低分子量麦谷蛋白D位点亚基可溶性的作用并未达到预期效果,但研究结果为SUMO标签增溶体系的建立和小麦低分子量麦谷蛋白亚基功能研究奠定了基础.

Exploration of Prokaryotic Expressing Solubilization System of the Gene XYGLuD3-LMWGS1 from Commom Wheat

In order to know further about the quality Function of XYGluD3-LMWGS1 and exploration of prokaryotic expressing solubilization system with SUMO1-tag,the coding XYGluD3-LMWGS1-noP(LnoP)were amplified from XYGluD3-LMWGS1 /pGEM-Teasy plasmid by PCR and constructed prokaryotic expression vector LnoP/pGEX-4T-1,LnoP-SUMO1/pET-41a( ),LnoP-SUMO1-N2/pET-41a( ),and LnoP-SUMO1/pET-32a( ) respectively.The eukaryotic expression vectors were transformed into E.coli BL21(DE3) pLysS or OrigamiB,and then the expression of fusion proteins GST-LnoP,GST-SUMO1-LnoP,SUMO1-LnoP,Trx-SUMO1-LnoP were induced by IPTG.Electrophoresis and Western blot were used to analyse the solubility of the expression of fusion proteins.Fusion protein GST-LnoP,GST-SUMO1-LnoP,SUMO1-LnoP,Trx-SUMO1-LnoP which induced by IPTG expressed successfully.GST-LnoP and GST-SUMO1-LnoP show no solubility.SUMO1-LnoP and Trx-SUMO1-LnoP show very small solubility.This result may illuminate the human SUMO1 work inefficiently in enhancing soluble protein expressing when it has been fused to the N-terminus of LnoP which is coded by XYGluD3-LMWGSI-noP gene in commom wheat.This is the first time SUMO-tag has been fused to the gene which is coded by low molecular weight protein subunits in commom wheat and expressed successfully.