title: High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.
abstract: Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.
authors.affiliation: Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Mlynská dolina, 842 15 Bratislava 4, Slovak Republic. Electronic address: levarski@fns.uniba.sk.
authors.forename: Zdenko
authors.initials: Z
authors.lastname: Levarski
authors.affiliation: Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Mlynská dolina, 842 15 Bratislava 4, Slovak Republic. Electronic address: soltysova@fns.uniba.sk.
authors.forename: Andrea
authors.initials: A
authors.lastname: Šoltýsová
authors.affiliation: Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Mlynská dolina, 842 15 Bratislava 4, Slovak Republic. Electronic address: krahulec@fns.uniba.sk.
authors.forename: Ján
authors.initials: J
authors.lastname: Krahulec
authors.affiliation: Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Mlynská dolina, 842 15 Bratislava 4, Slovak Republic. Electronic address: stuchlik@fns.uniba.sk.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Stuchlík
authors.affiliation: Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Mlynská dolina, 842 15 Bratislava 4, Slovak Republic. Electronic address: turna@fns.uniba.sk.
authors.forename: Ján
authors.initials: J
authors.lastname: Turňa
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics / Q000302:isolation & purification; D014169:Transformation, Bacterial
chemical_list: D011994:Recombinant Proteins; D019382:Human Growth Hormone; D013879:Thioredoxins
keywords: Escherichia coli; Human growth hormone; Nb2 assay; Recombinant protein