PRODUCTION OF SOLUBLE RECOMBINANT MYCOBACTERIUM TUBERCULOSIS AG85B BY ENDOGENOUS PROTEOLYTIC REMOVAL OF N-TERMINAL THIOREDOXIN IN E. СOLI

Abstract

Recombinant expression of Mycobacterium tuberculosis antigen 85B (Ag85B) in Escherichia coli is frequently limited by poor solubility and accumulation of the target protein in inclusion bodies. Fusion to thioredoxin (Trx) can improve solubility; however, subsequent removal of the fusion partner using commercial proteases adds an additional purification step and increases process complexity. Here, we developed an alternative approach in which optimization of cultivation conditions promoted both soluble expression and intracellular removal of the N-terminal Trx partner. A recombinant Trx-Ag85B-FLAG-His₆ construct was expressed in E. coli SHuffle T7, and the effects of medium composition, inducer concentration, cell density at induction, temperature, and cultivation time were evaluated. Under initial LB-medium conditions, the recombinant protein accumulated predominantly in the insoluble fraction as a full-length product of approximately 51 kDa, consistent with retention of the Trx fusion partner. Optimization using Super Broth-medium, 0.1 mM IPTG, induction at OD₆₀₀ 0.8–1.0, and cultivation at 16 °C substantially increased the soluble fraction. Prolonged incubation under these conditions resulted in a FLAG-positive product with a molecular mass below 35 kDa, consistent with removal of the N-terminal Trx and retention of the C-terminal Ag85B-FLAG-His₆ region. Addition of β-mercaptoethanol to the culture medium did not substantially affect accumulation of soluble protein. These findings support a model in which moderate expression kinetics facilitate soluble Ag85B folding, followed by endogenous intracellular proteolytic processing of the fusion protein. This strategy may simplify recombinant Ag85B production by reducing the need for an external protease-mediated tag-removal step.