Enhancement of the stability of Mycobacterium tuberculosis recombinant antigen expressed in Escherichia coli using cell lysis additives.

Enhancement of the stability of Mycobacterium tuberculosis recombinant antigen expressed in Escherichia coli using cell lysis additives.

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Highlights

We evaluated the effects of additives on the stability of Mtb antigens expressed in E. coli.

Degraded proteins were observed after incubation at 37 °C for 1 week.

Various additives, including sugars, stabilized recombinant Mtb proteins.

Recombinant Mtb antigen could be stabilized using chemical additives.

Abstract

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.

Keywords

Stabilizing additives
Mycobacterium tuberculosis
Escherichia coli
Recombinant mycobacterial antigen
Jawahar Raina

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