title: Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli.
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.
authors.affiliation: Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997 GSP, Moscow, Russia. marine@nmr.ru
authors.forename: Marine E
authors.initials: ME
authors.lastname: Gasparian
authors.forename: Valeriy G
authors.initials: VG
authors.lastname: Ostapchenko
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Schulga
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004228:Dithionitrobenzoic Acid / Q000032:analysis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000737:chemistry; D018793:Interleukin-13 / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D018793:Interleukin-13; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D007544:Isopropyl Thiogalactoside; D013879:Thioredoxins; D004815:Epidermal Growth Factor; C020520:thiobenzyl benzyloxycarbonyl-L-lysinate; C023519:glycyl-aspartyl-aspartyl-aspartyl-aspartyl-lysine-2-naphthylamide; D004228:Dithionitrobenzoic Acid; D004765:Enteropeptidase; D008239:Lysine