Cloning, prokaryotic expression and characterization of a protease from Thermococcus kodakarensis KOD1

• 1、College of Plant Science, Jinlin University, Changchun 130000

Abstract：In order to establish a foundation for further analysis of the structure and characterization of the protease, in this study, we constructed a recombinant Escherichia coli strain to produce protease TK0512 from Thermococcus kodakarensis KOD1. The recombinant enzyme was purified to characterize. The protease encoding gene TK0512 was cloned from genome DNA of Thermococcus kodakarensis KOD1. The amplified fragment was inserted into pET28a (+) to construct expression vector which was expressed in induced with isopropyl-β-D-thiogalactopyranoside. The recombinant protease was purified by affinity chromatography and their catalytic properties were characterized. A 705bp fragment of TK0512 was amplified from the KOD1 genomic DNA. SDS-PAGE showed TK0512 gene was expressed as a 28 kDa fusion protein with IPTG induction. The purified protease can decompose casein, BSA and even itself as a substrate. The optimum temperature of the enzyme activity is between 55-60 C, with the optimum pH value of 8.0-9.0. Its activity is significantly dependent on Zn2 +, while EDTA inhibits this activity. It can be concluded that this enzyme is a thermophilic metalloproteinase, which has great potential for application in industrial production.

Keywords： Thermophilic archaea metalloproteinase cloning prokaryotic expression enzyme assay