摘要
目的:表达和纯化人组氨酸磷酸酶PHP14蛋白,并寻找与其存在体外相互作用的蛋白。方法:PCR扩增PHP14的全长cDNA,并将其克隆至pGEX4T原核表达载体,构建重组表达质粒。用重组表达质粒转化大肠杆菌BL21菌株,经IPTG诱导后,使用Glutathione Sepharose 4B凝胶颗粒进行亲和纯化,通过SDS-PAGE电泳确定融合蛋白的表达。进行GST-Pulldown实验,寻找体外与PHP14相互作用的蛋白,并进行质谱鉴定。结果:在大肠杆菌BL21中成功了表达出了PHP14的可溶性融合蛋白;经Glutathione Sepharose4B凝胶颗粒纯化后,获得了纯化的GST-PHP14融合蛋白,GST-Pulldown实验和质谱鉴定结果发现PHP14与HSP90在体外存在相互作用。结论:实现了PHP14的原核表达和纯化,发现PHP14与HSP90在体外可能存在相互作用。
Objective： To express and purify human PHP14 protein and explore its interacting proteins in vitro. Methods： The cDNA sequence encoding PHP14 protein was amplified and subcloned into pGEX4T vector to construct the prokaryotic expression plasmid. The recombinant plasmid was transformed into E. coli BL21 strain and exogenous protein expression was induced by IPTG. After purification using Glutathione Sepharose 4B affinity chromatography, the GST-PHP14 fusion protein was separated by SDS-PAGE. GST-Pull down assay was performed to explore the interacting proteins in vitro and then subject to MALDI-TOF-MS identification. Results： GST-PHP14 fusion protein was expressed effectively in E. coli BL21 strain and successfully purified using Glutathione Sepharose 4B beads. Furthermore, GST-Pull down assay indicated that PHP14 could bind HSP90 in vitro. Conclusion： GST-PHP14 fusion protein was expressed and purified successfully and discoveried the interaction of PHP14 and HSP90 in vitro.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第7期7-11,共5页
China Biotechnology
基金
国家"973"计划(2006CB910100)
北京市自然科学基金(7051002)
北京市科学技术委员会基金(Y0204002040111)资助项目
