title: High level expression of human enteropeptidase light chain in Pichia pastoris.
abstract: Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.
authors: CPN spol. s r.o., Dolní Dobrouč 401, 56102 Dolní Dobrouč, Czech Republic. pepeliaev@contipro.cz
authors: Stanislav
authors: S
authors: Pepeliaev
authors: Ján
authors: J
authors: Krahulec
authors: Zbyněk
authors: Z
authors: Černý
authors: Jana
authors: J
authors: Jílková
authors: Marcela
authors: M
authors: Tlustá
authors: Jana
authors: J
authors: Dostálová
mesh_terms: D019149:Bioreactors; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004765:Enteropeptidase