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

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

Abstract

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

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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