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

• Published in: BioResearch open access Vol. 9; no. 1; pp. 86 - 93
• Main Authors: Kurniawati, Siti, Mertaniasih, Ni Made, Ato, Manabu, Tamura, Toshiki, Soedarsono, Soedarsono, Aulanni'am, Aulanni'am, Mori, Shigetarou, Maeda, Yumi, Mukai, Tetsu
• Format: Journal Article
• Language: English
• Published: United States 01.03.2020
• Subjects: expression; eccB5; ATPase; M. tuberculosis; cloning
• ISSN: 2164-7844; 2164-7860; 2164-7860
• DOI: 10.1089/biores.2019.0019

( ) is the causative agent of tuberculosis in human. One of the major virulence factors is early secretory antigenic target of 6-kDa (ESAT-6), and EccB protein encoded by is one of its components. EccB protein is a transmembrane protein in ESX-5 system. The aim of this study is to explore the characteristics of wild-type EccB and its mutant form N426I. We expressed the EccB protein by cloning the mutant and wild-type gene in ( ). We compared the protein structure of wild type and mutant form of EccB 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 EccB was successfully cloned and expressed using plasmid pCold I in DH5α and 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 EccB and its mutant. In conclusion, our study reveals successful cloning and protein expression of truncated form of eccB5 gene of . EccB 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.