NGAL蛋白Ni^(2+)-金属鏊合层析纯化及其鉴定  

Purification by Ni^(2+)-Metal Chelate Affinity Chromatography and Identification of NGAL Protein

作　　者：王朝阳[1] 许丽艳[1] 荣举[2] 李劲涛[1] 李恩民[2] 

机构地区：[1]汕头大学医学院肿瘤病理研究室,广东汕头515031 [2]汕头大学医学院生物化学与分子生物学教研室,广东汕头515031

出　　处：《癌变．畸变．突变》2004年第2期74-77,共4页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(No.399900069和No.30170428) ;广东省自然科学基金(No.010431) ;广东省高校自然科学研究基金(No.200033);广东省医学科研基金(No.A2001419);汕头大学研究与发展基金(No.L0004)

摘　　要：背景与目的 :通过对新癌基因NGAL的原核融合表达产物进行Ni2 +_金属鏊合层析纯化及其MALDI-TOF-MS鉴定 ,最后获得一定丰度与纯度的NGAL蛋白。 材料与方法 :将 pDsbA2.0 -NGAL融合表达载体转化大肠杆菌 ,进行IPTG诱导表达 ,SDS -PAGE分析表达产物的产量与可溶性 ,然后将表达产物进行Ni2 + _金属鏊合层析纯化及其MALDI-TOF-MS鉴定。 结果 :将 pDs bA2.0 -NGAL融合表达载体进行IPTG诱导表达 ,SDS-PAGE分析显示表达的NGAL融合蛋白产量高、可溶性好 ;对表达的NGAL蛋白进行Ni2 + _金属鏊合层析纯化后其纯度>95 %,MALDI-TOF-MS鉴定结果提示纯化后NGAL蛋白分子量与其理论分子量的误差仅为0.91‰。结论 :通过对新癌基因NGAL的原核融合表达产物进行Ni2 + _金属鏊合层析纯化及其MALDI-TOF-MS鉴定 ,最后确切获得了一定丰度电泳纯的NGAL蛋白 。BACKGROUND&AIM: To gain the NGAL protein with definite abundance and purity at last by the novel oncogene NGAL fusion expression in prokarote,purification by Ni 2+ _ metal chelate affinity chromatography and identification by MALDI-TOF-MS of its expression production. MATERIAL AND METHODS: pDsbA2.0-NGAL fusion expression vector was transformed to E.coli,induced to express with IPTG and productivity as well as soluˉbility of the expressed production were analyzed via SDS-PAGE.Then expressed production was purified by Ni 2+ _metal chelate affinity chromatography and identification byMALDI-TOF-MS. RESULTS: After induced expression of pDsbA2.0-NGAL fusion expression vector with IPTG,SDS-PAGE analysis presented the expressed NGAL fusion protein was high productivity with good solubility.Thepurity of expressed NGAL protein was more than95%after purified by Ni 2+ _metal chelate affinity chromatographyand identification by MALDI-TOF-MS indicated that the molecularweight error was only0.91‰between the purified NGAL protein and its theoretical one. CONCLUSION: By the novel oncogene NGAL fusion expression inprokarote,purification by Ni 2+ _metal chelate affinity chromatography and identification by MALDI-TOF-MS of its expression production,NGAL protein with definite abundance and electrophoresis purity was truely gained at last,which built up a good experimental material for the preparation of its antibody.

关 键 词：NGAL Ni^2+-金属鏊合层析 6×His MALDI-TOF-MS 

分 类 号：R730[医药卫生—肿瘤] Q503[医药卫生—临床医学]