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Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A

Paper ID Volume ID Publish Year Pages File Format Full-Text
22680 43369 2016 8 PDF Available
Title
Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A
Abstract

•Octa-histidine tagged hPGHS-2 was expressed in the yeast Pichia pastoris.•Compared to constitutive expression, methanol-induced expression yielded more hPGHS-2.•Bovine carboxypeptidase A was used to elute hPGHS-2 from the Ni-affinity resin.•3 mg of pure de-tagged hPGHS-2 was obtained from 1 l of P. pastoris culture.

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 1 l of yeast culture was approximately 3 mg. The protein purification process with simultaneous removal of the C-terminal polyhistidine tag could be easily applied for the affinity purification of other proteins.

Keywords
AA, arachidonic acid; AOX1, alcohol oxidase 1; boCPA, bovine carboxypeptidase A; CT, threshold cycle; GAP, glyceraldehyde 3-phosphate dehydrogenase; h, human; HIS4, histidinol dehydrogenase; qPCR, quantitative PCR; PGHS, prostaglandin H synthase; WCW, wet
First Page Preview
Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 231, 10 August 2016, Pages 224–231
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering