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High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris

2004; Elsevier BV; Volume: 108; Issue: 2 Linguagem: Inglês

10.1016/j.jbiotec.2003.11.004

1873-4863

Lisheng Peng, Xiaofen Zhong, Jingxing Ou, Suilan Zheng, Jian Liao, Lei Wang, Anlong Xu,

Enzyme Production and Characterization

Enterokinase (EC 3.4.21.9) is a serine proteinase with a specific digest sequence (Asp)4-Lys in the duodenum. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins. In this work, an active bovine enterokinase light chain (EKL) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris. The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied. The production of recombinant EKL by Muts strain was much higher than that by Mut+ strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EKL (rEKL) reached 350 mg l−1, which was 20-fold higher than that reported previously. The recombinant EKL was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EKL were obtained from 100 ml culture broth supernatant. The specific activity of purified rEKL was approximately 9000 u mg−1. To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EKL (EKL/His) was also expressed in P. pastoris, and this His-tagged EKL exhibited a similar enzymatic activity to the untagged EKL.