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.
Highlights
Tumor necrosis factor alpha (TNF-α) plays important and contradictory roles in the pathogenesis of many infectious diseases, including tuberculosis (TB) [1, 2]
On the other hand, uncontrolled TNF-α contributes to disease symptoms, tissue destruction, and organ pathology in TB and other chronic diseases [7]
Upon streaking on LB plate supplemented with antibiotics, bacterial transformants containing the cloned guinea pig TNFα gene were obtained
Summary
Tumor necrosis factor alpha (TNF-α) plays important and contradictory roles in the pathogenesis of many infectious diseases, including tuberculosis (TB) [1, 2]. On the other hand, uncontrolled TNF-α contributes to disease symptoms (e.g., fever and weight loss), tissue destruction, and organ pathology in TB and other chronic diseases [7]. Understanding these apparently contradictory functions of TNF-α will require the necessary reagents to study the molecule in both in vitro and in vivo studies in the small experimental animals of choice. Animal models such as mice, guinea pigs, rabbits, and monkeys are widely used in TB research [8]. Our laboratory has cloned and expressed several guinea pig cytokine and chemokine genes such as interleukin-8 (IL-8/CXCL-8) [10], regulated upon activation, normal Tcell expressed and secreted (RANTES/CCL5) [11], interferongamma (IFN-γ) [12], interleukin-4 (IL-4) [13], interleukin (IL-10) [14], interleukin-1beta (IL-1beta) [15], monocyte
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