Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

Brazilian Archives of Biology and Technology

Li-xi Niu¹  Jia-yue Li¹  Xue-xue Ji¹  Bin-sheng Yang¹ 

关键词: Human enterokinase light chain (hEKL);    Fusion expression;    Affinity purification;    Activity analysis;    Enzymatic cleavage;   

DOI  :  10.1590/S1516-8913201400094

来源: SciELO

【 摘 要 】

Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia colicodon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104U/mg.

【 授权许可】

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