Background: Mycobacteria tuberculosis (Mtb), the causative agent of tuberculosis, is a slow-growing bacterium. Expression in Escherichia coli is a widely used method for large-scale production of diagnostic antigenic recombinant proteins. Expression of Mtb antigen in E. coli offers a rapid and, inexpensive alternative to conventional protein synthesis from Mtb. The addition of stabilizing additives during cell lysis or storage of Mtb antigenic protein plays a vital role in enhancing antigen stability. In this study, we evaluated the effects of additives on the stability of Mtb antigens expressed in E. coli.
Methods: Immunodominant Mtb antigens, i.e., CFP-10, Rv3872, TB7.7, and TB9.7, were cloned, and recombinant proteins overexpressed in E. coli were gradually degraded in a time-dependent manner by incubation at 37 °C. Various stabilizing additives during storage or cell lysis before protein purification were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.
Results: CFP-10 and Rv3872 were mainly expressed in soluble form. The degraded form of the expressed protein after incubation at 37 °C was easily observed after 1 week. Increased stability was observed in a solution containing glycine for recombinant CFP-10 and Rv3872. TB9.7 was stable in a solution containing trehalose or mannitol. TB7.7 was stable in a solution containing sucrose, glycine, or polyethylene glycol.
Conclusion: Recombinant Mtb antigen stabilization using chemical additives inhibited protein degradation, leading to increased antigen stability and purification efficiency.
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