鸡溶菌酶和重组大肠杆菌融合蛋白(NADP-GDH)体外分子交联的初步研究

用PCR方法克隆的大肠杆菌NADP特异性谷氨酸脱氢酶 (NADP specificglutamatedehydrogenase ,NADP GDH)基因和其突变基因 ,插入表达载体pTrcHisC构建重组蛋白质表达体系 .经过IPTG诱导表达 ,用Talon固定化金属亲和层析树脂纯化出重组的天然和突变NADP GDH ,将它们和溶菌酶 (Lysozyme)通过热诱导去折叠方法进行体外蛋白质分子交联实验 ,在去折叠反应液中加入还原剂二硫苏糖醇 (DTT)后 ,不但没有分子间二硫键交联形成 ,同时也没有其他分子间共价键 (异肽键 )交联的形成 .另外 ,半胱氨酸残基定点突变后的NADP GDH重组蛋白质 ,无法参与形成分子间二硫键 ,实验证实经过热诱导去折叠后也没有分子间共价键 (异肽键 )交联 .这些结果进一步证明蛋白质分子间二硫键的形成能够促进蛋白质其他分子间共价键 (异肽键 )的形成 .

Protein Cross-linking During Thermal Unfolding of Chicken Lysozyme and Recombinant Escherichia coli Protein in Vivo

The Escherichia coli NADP-specific glutamate dehydrogenase gene(gdhA) and Mutant NADP-specific glutamate dehydrogenase gene,cloned by PCR,were iserted into plasmid pTrcHisC for over expression and induced by IPTG,the recombinant proteins were purified by using Talon metal affinity resin.Thermal unfolding of recombinant Escherichia coli NADP-specific glutamate dehydrogenase,Mutant NADP-specific glutamate dehydrogenase,and Lysozyme lead to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis,in addition,heterogenerous proteins also cross-linked.Preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide bond cross-links,because no dimers/oligomers were detected when the unfolding solution contained the reducing agent dithiothreitol.Furthermore,the Cys334Ser point mutation in ribonucleic acid enzyme HII abrogated its ability to cross-link into homodimers.All these suggested that protein cross-linking can be accomplished in three concerted steps:(i) changes in protein conformation,(ii) formation of inter-molecule disulfide bonds,and (iii) formation of inter-molecule isopetide cross-linking.