title: Production of a recombinant bovine enterokinase catalytic subunit in the methylotrophic yeast Pichia pastoris.
abstract: We describe the heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris. A highly active protein is secreted and glycosylated, and it has the native amino-terminus of EKL. The cDNA encoding EKL was cloned with the KEX2 protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevisiae. The secreted EKL was easily purified from the few native proteins found in the P. pastoris fermentation supernatant, using ion exchange and affinity chromatography. The yield of the purified EKL was 6.3 mg per liter of fermentation culture. This is significantly higher than previous reports of expressions in E. coli and COS cells. The ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino-terminal residues of recombinant proteins. Its application is demonstrated by the removal of thioredoxin (TrxA), and polyhistidine fusion partners from proteins of interest.
authors.affiliation: Invitrogen Corporation, San Diego, CA 92121, USA. lvozza@invitrogen.com
authors.forename: L A
authors.initials: LA
authors.lastname: Vozza
authors.forename: L
authors.initials: L
authors.lastname: Wittwer
authors.forename: D R
authors.initials: DR
authors.lastname: Higgins
authors.forename: T J
authors.initials: TJ
authors.lastname: Purcell
authors.forename: M
authors.initials: M
authors.lastname: Bergseid
authors.forename: L A
authors.initials: LA
authors.lastname: Collins-Racie
authors.forename: E R
authors.initials: ER
authors.lastname: LaVallie
authors.forename: J P
authors.initials: JP
authors.lastname: Hoeffler
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D004765:Enteropeptidase / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli; D005822:Genetic Vectors; D006639:Histidine; D010446:Peptide Fragments / Q000096:biosynthesis*; D010455:Peptides / Q000378:metabolism; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D013879:Thioredoxins / Q000096:biosynthesis
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D013879:Thioredoxins; D004765:Enteropeptidase