国家自然科学基金(39770307)及教育部骨干教师资助计划资助.
This work was supported by grants from the National Natural Science of Foundation of China(39770307)and Key Member Award from the Ministry of Education.
为将αB-晶状体蛋白(αB-crystallin, αB-C)导入心肌细胞,采用蛋白质工程技术将人αB-晶状体蛋白全长cDNA基因克隆至具有细胞膜通透能力的膜移位序列(membrane-translocating sequence,MTS)的碱基顺序的下游,在大肠杆菌中表达谷胱甘肽5-转移酶(GST)-MTS-αB-C融合蛋白.用谷胱甘肽-Sepharose4B亲和层析分离表达产物,用Xa因子将其中GST切除,并通过阴离子交换层析纯化MTS-αB-C.纯化的MTS-αB-C在SDS-聚丙烯酰胺凝胶电泳(PAGE)上呈单一条带,分子质量23 ku;免疫印迹显示GST-MTS-αB-C与MTS-αB-C均能为人αB-C抗体识别而在相应位置出现清晰条带.GST-MTS-αB-C与MTS-αB-C均有分子伴侣活性.用异硫氰荧光素(FITC)标记的MTS-αB-C与心肌细胞共培养后,在荧光显微镜下观察到其进入了心肌细胞.
In order to deliver αB-crystallin (αB-C) into cardiomyocytes,the full-length cDNA fragment encoding the human αB-crystallin was cloned into the bacterial expression vector pGEX-MTS containing membrane-translocating sequence(MTS) which could mediate intracellular delivery of peptides and expressed as a fusion protein coupled to glutathione S-transferase(GST).After glutathione affinity chromatography and cleaved from GST by factor Xa,the recombinant MTS-αB-C was separated from GST and factor Xa by anion exchange chromatography.Recombinant MTS-αB-C was characterized by SDS-PAGE and Western immunoblot analysis.The purified MTS-αB-C migrated on SDS-PAGE as a single band to an apparent molecular mass (23 ku) that corresponded to total native αB-C and MTS,and was recognized on Western immunoblot by anti-human αB-crystallin antibody. Both MTS-αB-C and GST- MTS-αB-C displayed chaperone like function by disaggregating the denatured and aggregated actin induced by H2O2 treatment in an ATP-containing buffer at 37℃.It was observed under fluorescence microscope that FITC-labeled MTS-αB-C had gone into neonatal rat cardiomyocytes by MTS mediation after the cells were incubated with the MTS-αB-C for 8 hours.
蒋磊,刘双,袁灿,肖卫民,王慷慨,尢家(马录),肖献忠.采用蛋白质工程技术将αB-晶状体蛋白导入心肌细胞的初步研究[J].生物化学与生物物理进展,2002,29(2):283-287
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