特异识别HIV-1序列的锌指蛋白的表达纯化及其在Biacore CM-5芯片上的固定

目的:为了更好地利用Biacore 3000研究锌指与核酸的相互作用,将特异性识别HIV-15′端一段保守序列的三锌指蛋白固定在CM-5芯片上。方法:将特异性识别HIV-15′端一段保守序列5′-CTGTGTTTG-3′的三锌指基因克隆到表达载体pET-22b( )中,转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达重组三锌指蛋白,超声碎菌进行SDS-PAGE分析;包涵体形式的表达产物用盐酸胍溶解后,经一步凝胶柱复性并纯化;随后摸索适宜固定的pH值并通过化学方法进行固定。结果:表达的重组蛋白主要以包涵体形式存在于超声沉淀中,纯化及柱复性后的蛋白纯度为98.8%,并在CM-5芯片上成功固定。结论:本研究为利用Biacore实时定量研究锌指蛋白与其识别DNA的相互作用进行了尝试。

Expression and Purification of a Zinc Finger Specific to HIV-1 5'-Leader Sequence and its Immobilization on Sensor Chip CM-5

Objective:To immobilize a three zinc finger protein that specific to a 9 bp sequence in HIV-1 on sensor chip CM-5.Methods:A three zinc finger gene which specific to a 9 bp sequence 5'-CTGTGTTTG-3' in HIV-1 5'-leader sequence has been cloned,then was inserted into a prokaryotic expression vector named pET-22b( ).The ecombinant plasmid was transformed into E.coli BL21(DE3)and induced with IPTG.Inclusion bodies were refolded and purified by gel-filtration chromatography using superdex 75.Then the purified and concentrated zinc finger protein were immobilized on sensor chip CM-5 using amine coupling chemistry methods.Results:The zinc finger protein was expressed sucessfully in form of inclusion body,and the purification rate of the on-column refolding zinc finger protein was up 98.8%.Conclusion:The purified protein was immobilized on a sensor chip CM5 for further Biacore analysis.