Cloning Of Three Different Protease Genes From Natrinema-IM-1 And Construction Of The Expression Vectors

Archaea is an organism distinguished from eukaryotes and bacteria.It can be divided into Crenarchaeota and Euryarchaeota,among which halophilic archaea is a branch of Euryarchaeota and can exist in salt concentrations above 30%.Protease is a type of enzyme that catalyzes the hydrolysis of proteins and acts primarily on peptide bonds between amino acids.Protease has a wide range of applications in many fields such as pharmaceutical and chemical industry,food industry,and wine industry and textiles.In order to study archaeal-sourced proteases,the genome sequence of Natrinema-sp-J7-2in GenBank has been analyzed and specific primers has been designed,and three different protease genes have been amplified from the same genus Natrinma-IM-1 which conserved in our lab.The successfully cloned three different protease genes of Natrinema-IM-1 have been inserted into the expression vector pET-28a(+),and three recombinant expression vectors were successfully constructed and named as pET-28a-MDP,pET-28a-SP,pET-28a-P.This study laid the foundation for the expression of serine protease,protease and metal-dependent protease from Natrinema-IM-1in E.coli,and for the enzyme activity analysis.This study also provided experimental data and theoretical basis for further development and utilization of halophilic archaeal proteases.