Cloning and Protein Expression of eccB5 Gene in ESX-5 System from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis in human. One of the major M. tuberculosis virulence factors is early secretory antigenic target of 6-kDa (ESAT-6), and EccB₅ protein encoded by eccB₅ is one of its components. EccB₅ protein is a transmembrane protein in ESX-5 system. The aim of this study is to explore the characteristics of wild-type EccB₅ and its mutant form N426I. We expressed the EccB₅ protein by cloning the mutant and wild-type eccB₅ gene in Escherichia coli (E. coli). We compared the protein structure of wild type and mutant form of EccB₅ and found changes in structure around Asn426 (loop structure) in wild type and around Ile426 (β-strand) in the mutant. The truncated recombinant protein of EccB₅ was successfully cloned and expressed using plasmid pCold I in E. coli DH5α and E. coli strain Rosetta-gami B (DE3) and purified as a 38.6 kDa protein by using the affinity column. There was no detectable adenosine triphosphatase activity in truncated forms of EccB₅ and its mutant. In conclusion, our study reveals successful cloning and protein expression of truncated form of eccB5 gene of M. tuberculosis. EccB₅ protein in ESX-5 system may be an important membrane component involved in the transport machinery of type VII secretion system, which is essential for growth and virulence.