The nucleotide sequence encoding bovine enterokinase light chain (EK) from Chinese northern yellow bovine was isolated. Two single-nucleotide mutations, namely, C245G and A528T were identified. The gene encoding the Pro82Arg/Glu176Asp variant of known bovine EK was fused with glutathione S-transferase and overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with IPTG and glucose. Effective fusion protein purification, refolding, auto-catalytic cleavage and mature EK recovery were described. The specific activity of the purified EK was determined as 110+/- 10 U/mg, which was comparable to a specific activity of > or =20 U/mg of the E. coli expressed EK sample provided by Sigma (Cat. No. E4906). This procedure produced approximately 53 mg of EK per 500 mL of cell culture, which was much higher than previous reports, thus providing a basis for large-scale production of EK and for further applications in biotechnology.