title: Prokaryotic overexpression of TEV-rhGH and characterization of its polyclonal antibody.
abstract: Recombinant protein technology represents one of the best solutions to achieve rapid, efficient, and cost-effective protein expression and purification of therapeutic proteins. Growth hormone (GH) is an excellent example of these proteins used in the therapy of hormone deficiencies. In this work, a plasmid, pRSET-TEV-rhGH, has been constructed to overexpress recombinant human GH (rhGH) by cloning its gene downstream of an N-terminal 6 × His-tagged polypeptide (43 aa) in the T7 promoter-plasmid pRSET. This polypeptide was cleavable by means of the integrated recognition site for the tobaccos etch virus (TEV) protease, resulting in an rhGH protein at an exact length and sequence. After IPTG induction, this plasmid effectively expressed TEV-rhGH protein (27 kDa) in the cytoplasm of Escherichia coli, which accumulated in the form of inclusion bodies. The 6 × His-tagged protein, with a yield of ~150 mg/L of culture, was purified from the cell extract using metal affinity chromatography, as shown after SDS-PAGE blue staining, and was confirmed by immunoblotting using specific commercial monoclonal antibodies. In order to detect TEV-rhGH, in ELISA and immunoblotting, specific polyclonal antibody, with high titer (~10⁻⁵ fold dilution), was produced in a rabbit and purified using affinity chromatography. Preliminary tests have proved that TEV-rhGH protein and its specific purified IgG antibody could provide valuable tools for rhGH productive and diagnostic purposes.
authors.affiliation: Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P.O. Box 6091, Damascus, Syria.
authors.forename: Hossam
authors.initials: H
authors.lastname: Murad
authors.affiliation: Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P.O. Box 6091, Damascus, Syria.
authors.forename: Bouthina
authors.initials: B
authors.lastname: Ali
authors.affiliation: Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P.O. Box 6091, Damascus, Syria.
authors.forename: Rima
authors.initials: R
authors.lastname: Makeya
authors.affiliation: Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P.O. Box 6091, Damascus, Syria. Electronic address: ascientific@aec.org.sy.
authors.forename: Abdul Qader
authors.initials: AQ
authors.lastname: Abbady
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D019382:Human Growth Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D010450:Endopeptidases; C099488:TEV protease
keywords: Affinity purification; Polyclonal antibody; Protein expression; Recombinant growth hormone; TEV protease