High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris.
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 (EK(L)) 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 EK(L) by Mut(s) strain was much higher than that by Mut+ strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK(L) (rEK(L)) reached 350 mg l(-1), which was 20-fold higher than that reported previously. The recombinant EK(L) was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK(L) were obtained from 100 ml culture broth supernatant. The specific activity of purified rEK(L) was approximately 9000 u mg(-1). To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EK(L) (EK(L)/His) was also expressed in P. pastoris, and this His-tagged EK(L) exhibited a similar enzymatic activity to the untagged EK(L).
Peng
Lisheng
L
The Open Laboratory for Marine Functional Genomics of State High-Tech Development, Department of Biochemistry, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, PR China.
Zhong
Xiaofen
X
Ou
Jingxing
J
Zheng
Suilan
S
Liao
Jian
J
Wang
Lei
L
Xu
Anlong
A
D000818:Animals; D002244:Carbon / Q000378:metabolism; D002417:Cattle; D004765:Enteropeptidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D002244:Carbon; D004765:Enteropeptidase; D000432:Methanol