Expression of soluble recombinant Mycobacterium tuberculosis early secreted antigen target 6 (ESAT6) using cold-inducible expression system: A prospect for Mycobacterium tuberculosis skin test detection

Tuberculosis (TB) remains a global health issue. One of the major challenge in TB control are over diagnose and underdiagnosed which consequently would lead to increase burden of disease. Therefore, there is urgency for developing a specific and sensitive diagnostic tool. Early secreted antigen target 6 (ESAT6) has been studied extensively as a prospective tuberculosis diagnostic tool. It has the ability to distinguish between TB infection and non TB infection (infection due to non-Tuberculosis mycobacterium and previous BCG vaccinated individual). Production of the antigen in a high-level soluble form is required to provide material for skin test as a more accurate diagnostic tool for tuberculosis. In this research, ESAT6 recombinant protein was over expressed primarily as a soluble protein in high-level using combination of E. coli BL21codon Plus RIPL Strain with cold-inducible expression system. Since the BL21 codon plus (DE3)RIPL cells contain extra copies of argU, ileY, leuW, and proL tRNAs genes, the expression of pLDH which contains 11% of the rare tRNAs was efficiently rescued. Moreover, the use of cold temperature (15 o C) has reduced cell growth, slowed down transcription and translation rate, and reduced the strength of hydrophobic interactions that contribute to the protein misfolding. This research provides a convenience method for generation of high quality and quantity of recombinant antigen.