【作者】 周霞；

【导师】 高音；

【作者基本信息】 首都师范大学 ， 生物学遗传学， 2003， 硕士

【摘要】 蛋白质分子间的交联是普遍存在的现象。它是多种生理和病理过程的共同特征。然而，由蛋白质的单体或功能形式转变为交联的二聚体或多聚体的分子机理还不太清楚。溶菌酶、核糖核酸酶A和蛋白质二硫键异构酶的体外实验显示，蛋白质交联可经三步完成：1)蛋白质构象包括二级结构改变；2)形成分子间二硫键；3)形成分子间异肽键。为了进一步探索蛋白质交联途径的三步假说的可行性，以及交联能否在异源肽链间发生，本文以人肽基脯氨酰顺反异构酶cyclophilin为模型展开了深入地研究。用RT-PCR方法从中国人胚肝中克隆了hPPI cyclophilin cDNA基因，并构建pTrcHisC-PPI重组质粒在大肠杆菌中高效表达，应用（His）₆亲和标签的固定化金属亲和层析技术纯化出了hPPI cyclophilin蛋白。将hPPI cyclophilin和lysozyme蛋白进行热变性交联实验，结果显示：在热变性的临界温度时，同源和异源的肽链间都有交联的二聚体和多聚体形成；而低温条件下，却没有交联出现；在热变性样品中加入二硫键还原剂β-巯基乙醇后，也检测不到任何交联。很明显，只有当蛋白质的二级结构改变到一定程度，才会发生交联；而且二硫键交联的形成对异肽键交联的形成起促进作用。这些都为蛋白质交联途径的三步假说提供了一些证据。更多还原

【Abstract】 Protein interchain cross-linking is a common phenomenon. It has been implicated in several physiological and pathological processes. However the molecular mechanisms for converting monomeric/functional forms of proteins into cross-linked dimer/oligomer forms are not well understood. Experiments with lysozyme, ribonuclease A and protein disulfide isomerase in vitro show protein cross-linking can be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links. For further exploring if the three-step hypothesis of protein cross-linking is right, and if cross-linking can happen hi heterogeneous peptides, the investigation is extended with human peptidylprolyl-cis-trans-isomerase cyclophilin (hPPI cyclophilin). hPPI cyclophilin cDNA was cloned by RT-PCR from Chinese human fetal liver, and inserted into plasmid pTrcHisC for overexpression in E. coli. Then it was purified by Immobilized Metal Affinity Chromatography with the help of 6*His affinity tag. At last, hPPI cyclophilin and lysozyme were mixed during thermal unfolding. The observation revealed that heterodimer/oligomer formation was as common as homodimer/oligomer formation in the midpoint temperature of thermal denaturation. However no cross-linked dimer/oligomer appeared in low temperature. If the thermal unfolding solution contained the reducing agent P -Mercaptoethanol, no cross-linking was detected. It was apparent that cross-linking can’t happen until the protein secondary structure had changed to a certain extent, and that preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links. All these results confirmed the three-step hypothesis of protein cross-linking.更多还原