title: Molecular and biochemical characterization of recombinant guinea pig tumor necrosis factor-alpha.
abstract: Tumor necrosis factor alpha (TNF-α) is a cytokine which plays opposing roles in the context of infectious disease pathogenesis. TNF-α is essential for the development of a protective immune response to some pathogens, for example, Mycobacterium tuberculosis, by synergizing with other cytokines. However, exorbitant or uncontrolled TNF-α activity may also drive pathology and disease symptoms in many infectious diseases. In order to elucidate the beneficial and detrimental roles of TNF-α in tuberculosis (TB) and other diseases for which the guinea pig is the small animal model of choice, recombinant guinea pig (rgp)TNF-α has been produced using prokaryotic expression systems. However, it is unknown whether posttranslational modifications which cannot be made in the prokaryotic expression systems may be important for rgpTNF-α structure and function. Therefore, we carried out a comparative study by expressing rgpTNF-α in prokaryotic and eukaryotic expression systems and analyzed the eukaryotic-expressed rgpTNF-α for the presence of posttranslational modifications by subjecting it to NanoLC-MS/MS. We conclude that the eukaryotic-expressed rgpTNF-α lacks posttranslational modifications, and we found no significant difference in terms of the biological activity between prokaryotic- and eukaryotic-expressed rgpTNF-α. Taken together, results from our study show that a prokaryotic expression system can be used for generating large amounts of rgpTNF-α without concern for the biological integrity.
authors.affiliation: Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA ; Department of Biotechnology, Vignan's University, Guntur 522213, India.
authors.forename: Vijaya R
authors.initials: VR
authors.lastname: Dirisala
authors.affiliation: Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.
authors.forename: Amminikutty
authors.initials: A
authors.lastname: Jeevan
authors.affiliation: Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.
authors.forename: Lan H
authors.initials: LH
authors.lastname: Ly
authors.affiliation: Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.
authors.forename: David N
authors.initials: DN
authors.lastname: McMurray
mesh_terms: D000818:Animals; D016207:Cytokines / Q000378:metabolism; D006168:Guinea Pigs; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D016207:Cytokines; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha