title: Optimization of conditions for expression of recombinant interferon-γ in E.coli.
abstract: Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.
authors.affiliation: Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, New Delhi, 110054, India, meetulkumar@gmail.com.
authors.forename: Meetul
authors.initials: M
authors.lastname: Kumar
authors.forename: Mrinalini
authors.initials: M
authors.lastname: Singh
authors.forename: Shashi Bala
authors.initials: SB
authors.lastname: Singh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D011994:Recombinant Proteins; D007371:Interferon-gamma