Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation

A. Jeevan, C. T. McFarland, Teizo Yoshimura, T. Skwor, H. Cho, T. Lasco, D. N. McMurray

Research output: Contribution to journalArticle

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Abstract

Gamma Interferon (IFN-γ) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-γ (gpIFN-γ) and reported that BCG vaccination induced a significant increase in the IFN-γ mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-γ mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-γ with a histidine tag at the N terminus (His-tagged rgpIFN-γ) in Escherichia coli. rgpIFN-γ was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-γ. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-γ was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-γ did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-γ induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-γ has been successfully produced and characterized in our laboratory. The study of rgpIFN-γ will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

Original languageEnglish
Pages (from-to)213-224
Number of pages12
JournalInfection and Immunity
Volume74
Issue number1
DOIs
Publication statusPublished - Jan 2006
Externally publishedYes

Fingerprint

Macrophage Activation
Interferon-gamma
Guinea Pigs
Mycobacterium
Interferons
Peritoneal Macrophages
Mycobacterium bovis
Recombinant Proteins
Vaccination
Encephalomyocarditis virus
Messenger RNA
Protein Sequence Analysis
Major Histocompatibility Complex
Pulmonary Tuberculosis
Mycobacterium tuberculosis
Histidine
Hydrogen Peroxide
Antiviral Agents
Nitric Oxide
Complementary DNA

ASJC Scopus subject areas

  • Immunology

Cite this

Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation. / Jeevan, A.; McFarland, C. T.; Yoshimura, Teizo; Skwor, T.; Cho, H.; Lasco, T.; McMurray, D. N.

In: Infection and Immunity, Vol. 74, No. 1, 01.2006, p. 213-224.

Research output: Contribution to journalArticle

Jeevan, A. ; McFarland, C. T. ; Yoshimura, Teizo ; Skwor, T. ; Cho, H. ; Lasco, T. ; McMurray, D. N. / Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation. In: Infection and Immunity. 2006 ; Vol. 74, No. 1. pp. 213-224.
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