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Expression, Purification, and Characterization of Human Enteropeptidase Catalytic Subunit in Escherichia Coli

Expression, Purification, and Characterization of Human Enteropeptidase Catalytic Subunit in Escherichia Coli

Marine E Gasparian, Valeriy G Ostapchenko, Alexey A Schulga, Dmitry A Dolgikh, Mikhail P Kirpichnikov

Protein Expr Purif. 2003 Sep;31(1):133-9.

PMID: 12963350

Abstract:

Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP70023028	Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide		70023-02-8	Price
IAR42414878	Thioredoxin human			Price

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