Molecular and biochemical characterization of recombinant guinea pig tumor necrosis factor-alpha.

ABSTRACT: Tumor necrosis factor alpha (TNF-?) is a cytokine which plays opposing roles in the context of infectious disease pathogenesis. TNF-? 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-? activity may also drive pathology and disease symptoms in many infectious diseases. In order to elucidate the beneficial and detrimental roles of TNF-? in tuberculosis (TB) and other diseases for which the guinea pig is the small animal model of choice, recombinant guinea pig (rgp)TNF-? 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-? structure and function. Therefore, we carried out a comparative study by expressing rgpTNF-? in prokaryotic and eukaryotic expression systems and analyzed the eukaryotic-expressed rgpTNF-? for the presence of posttranslational modifications by subjecting it to NanoLC-MS/MS. We conclude that the eukaryotic-expressed rgpTNF-? lacks posttranslational modifications, and we found no significant difference in terms of the biological activity between prokaryotic- and eukaryotic-expressed rgpTNF-?. Taken together, results from our study show that a prokaryotic expression system can be used for generating large amounts of rgpTNF-? without concern for the biological integrity.

SUBMITTER: Dirisala VR 

PROVIDER: S-EPMC4427127 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature