Cloning, expression and purification of human and bovine Enterokinase light chain with Cherry tag and their activity comparison
Mahammad Azhar1*, R Somashekhar2
1Department of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad (India) 2Department of Biotechnology, Azyme Biosciences, Bangalore (India) *Corresponding Author Mail ID: ssrajur@gmail.com (R Somashekhar). Contact No. +91 9844787062
ABSTRACT
Enterokinase (E.C 3.4.21.9) is a serine protease, known for its specific cleavage after Asp-Asp-Asp-Asp- Lys .Enterokinase (EK) is a widely used tool for in vitro cleavage of recombinant fusion proteins produced by bacterial expression system. So far EK produced in bacterial system mainly expressing as inactive inclusion bodies, which requires an extensive denature and refolding process with low active protein. cDNA encoding the Catalytic Subunit of human Enterokinas was isolated from human dedunum complimentary DNA (cDNA) library (Gene bank accession No. KF688184). cDNA Encoding the Catalytic Subunit of Bovine EnterokinasBovine isolated from a Bos taurus indicus (Gene bank accession No. KC756844) both cDNA were cloned in psCherry1 vector between EcoR1, Xho1 restriction sites, fused to cherry tag. Human and bovine enterokinase fused with Cherry tag in pS cherry1 expression vectors were expressed in E. Coli Rosetta strain. Expressed human and bovine enterokinase light chains with cherry tag, most of the product existed in soluble form. After purification and dialysis auto cleave was observed.14mg/Liter pure active bovine enterokinase light chain protein obtained, without involving any denaturing and refolding process. Likewise 16mg/Liter human enterokinase light chain was purified. Recombinant bovine enterokinase light chain showing Km value 0.83± 0.03 mM and Kcat 25 Sec-1 for its speific fluorometric substrate Gly- (Asp)4- Lys-β-naphthylamide. In similar experimental conditions recombinant humane enterokinase light chain showing Km value 0.085± 0.001 mM and Kcat 118 Sec-1.