Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli.
Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase.
Huang
Lei
L
Institute of Bioengineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, PR China.
Ruan
Hong
H
Gu
Weiyan
W
Xu
Zhinan
Z
Cen
Peilin
P
Fan
Limei
L
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009842:Oligopeptides / Q000737:chemistry; D019897:Periplasm / Q000737:chemistry; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D014169:Transformation, Bacterial / Q000235:genetics
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D004765:Enteropeptidase