Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A.
Vertebrate prostaglandin H synthases (PGHSs) are membrane-bound disulphide-containing hemoglycoproteins. Therefore, eukaryotic expression systems are required for the production of recombinant PGHSs. Recently we announced the expression of human PGHS-2 (hPGHS-2) in the yeast Pichia pastoris. Here we report improved production of hPGHS-2 in P. pastoris and a convenient method for the purification and de-tagging of the protein. An affinity tag comprised of a proline, a glycine and eight histidines was introduced into the C-terminal end of hPGHS-2. The tagged hPGHS-2 was expressed intracellularly in P. pastoris under the control of a constitutive or methanol-inducible promoter. Compared to constitutive expression, methanol-induced expression yielded approximately four times more protein. The analysis of high and low gene copy number recombinants revealed a positive correlation between the gene copy number and the expression level of hPGHS-2. The recombinant hPGHS-2 was purified using immobilised metal ion affinity chromatography. A novel elution method, treatment of the affinity resin with bovine carboxypeptidase A, was employed. The yield of pure de-tagged hPGHS-2 from 1l of yeast culture was approximately 3mg. The protein purification process with simultaneous removal of the C-terminal polyhistidine tag could be easily applied for the affinity purification of other proteins.
Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
Kaia
K
Kukk
Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia. Electronic address: nigulas.samel@ttu.ee.
Nigulas
N
Samel
D000818:Animals; D043422:Carboxypeptidases A / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018628:Gene Dosage / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D051546:Cyclooxygenase 2; D043422:Carboxypeptidases A
Carboxypeptidase; Pichia pastoris; Prostaglandin H synthase; Protein purification; Recombinant expression; Tag removal