重组内肽酶AspN的原核表达纯化和活性鉴定  

Cloning,Expression,Purification and Characterization of Soluble Recombinant Endoproteainase AspN

作　　者：郑娟[1,2] 郭怀祖[2,3] 李晶[2,3] 段树燕 赵自叶 张大鹏[2,3] 郭尚敬[1] 王皓[2,3] 

机构地区：[1]聊城大学药学院,聊城252000 [2]抗体药物与靶向治疗国家重点实验室,上海201203 [3]第二军医大学肿瘤研究所,上海200433

出　　处：《生物技术通报》2015年第1期186-192,共7页Biotechnology Bulletin

摘　　要：蛋白内肽酶AspN是一种锌金属内切肽酶,能选择性切割天冬氨酸的N端肽键,广泛应用于蛋白的多肽制备及质量肽图谱鉴定。目前内肽酶AspN来源于细菌分泌,产量低,制备困难,成本高,极大地限制了该酶的应用。将脑膜脓毒性黄杆菌分泌的蛋白内肽酶Asp N所对应的基因克隆入表达载体p ET32a,导入E.coli BL21(DE3),首次运用原核表达系统进行可溶性融合表达,亲和层析对重组蛋白进行纯化。用HPLC、SDS-PAGE和荧光底物Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe(NO2)-Tyr-Asp对重组酶进行酶活鉴定。结果表明重组的内肽酶Asp N具有与标准品基本一致的酶切活性,能够较好地应用于生物和制药领域。Endoproteinase AspN（flavastacin）is a zinc metalloendopeptidase which can selectively cleave peptide bonds N-terminal to aspartic acid residues, also it is one of most widely used proteolytic enzyme used for protein digestion prior to MS or HPLC analysis. The natural endoproteinase AspN was extract from the bacteria,Elizabethkingia meningoseptica. Some factors including low yields, difficulty in purification and high cost, greatly limit the application of the enzyme. Target gene was cloned into an pET32a expression vector and transformed into E.coli BL21（DE3）. This is the first report for the soluble expression of AspN-Trx in prokaryotic expression system and a poly-histidine tag enabled purification by Ni affinity chromatography. The activity of the recombinant endopeptidase AspN was identified by HPLC, SDS-PAGE and the fluorogenic substrate Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe（NO2）-Tyr-Asp. The results showed that the recombinant endopeptidase AspN was consistent with the standard enzyme and can be used widely.

关 键 词：重组内肽酶AspN 原核表达 纯化 活性鉴定 

分 类 号：Q78[生物学—分子生物学]