重组内肽酶AspN的原核表达纯化和活性鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2015.01.028

生物技术通报 ›› 2015, Vol. 31 ›› Issue (1): 186-192.doi: 10.13560/j.cnki.biotech.bull.1985.2015.01.028

重组内肽酶AspN的原核表达纯化和活性鉴定

郑娟^1，2 郭怀祖^2，3 李晶^2，3 段树燕^1，2 赵自叶^1，2 张大鹏^2，3 郭尚敬王皓^2，3

（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）

收稿日期:2014-07-04 出版日期:2015-01-09 发布日期:2015-01-10
作者简介:郑娟，女，研究生，研究方向：分子细胞生物学；E-mail：zhengjuan2006400741@126.com

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

Zheng Juan^1，2, Guo Huaizu^2，3, Li Jing^2，3, Duan Shuyan^1，2 , Zhao Ziye^1，2, Zhang Dapeng^2，3, Guo Shangjing, Wang Hao^2，3

（1. School of Pharmacy of Liaocheng University，Liaocheng 252000；2. State Key Laboratory of Antibody Medicine and Targeted Therapy，Shanghai 201203；3. Second Military Medical University，Shanghai 200433）

Received:2014-07-04 Published:2015-01-09 Online:2015-01-10

摘要/Abstract

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AspN所对应的基因克隆入表达载体pET32a，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AspN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。

关键词: 重组内肽酶AspN, 原核表达, 纯化, 活性鉴定

Abstract: 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.

Key words: recombinant endoproteinase AspN, prokaryotic expression, purification, activity assay

郑娟,郭怀祖,李晶,段树燕,赵自叶,张大鹏,郭尚敬,王皓. 重组内肽酶AspN的原核表达纯化和活性鉴定[J]. 生物技术通报, 2015, 31(1): 186-192.

Zheng Juan, Guo Huaizu, Li Jing, Duan Shuyan , Zhao Ziye, Zhang Dapeng, Guo Shangjing, Wang Hao. Cloning，Expression，Purification and Characterization of Soluble Recombinant Endoproteainase AspN[J]. Biotechnology Bulletin, 2015, 31(1): 186-192.

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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

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