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

Siti Kurniawati, Ni Made Mertaniasih, Manabu Ato, Toshiki Tamura, Soedarsono Soedarsono, Aulanni'am Aulanni'am, Shigetarou Mori, Yumi Maeda, Tetsu Mukai
BioResearch Open Access 2020, 9 (1): 86-93
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.

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