Kurniawati Siti, Mertaniasih Ni Made, Ato Manabu, Tamura Toshiki, Soedarsono Soedarsono, Aulanni'am Aulanni'am, Mori Shigetarou, Maeda Yumi, Mukai Tetsu
Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia.
Department of Clinical Microbiology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia.
Biores Open Access. 2020 Mar 31;9(1):86-93. doi: 10.1089/biores.2019.0019. eCollection 2020.
() 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.
（）是人类结核病的病原体。主要毒力因子之一是6 kDa早期分泌性抗原靶标（ESAT-6），而由（）编码的EccB蛋白是其组成成分之一。EccB蛋白是ESX-5系统中的一种跨膜蛋白。本研究的目的是探索野生型EccB及其突变形式N426I的特征。我们通过在（）中克隆突变型和野生型（）基因来表达EccB蛋白。我们比较了EccB野生型和突变型的蛋白质结构，发现野生型中Asn426周围（环结构）和突变型中Ile426周围（β链）的结构发生了变化。使用质粒pCold I在DH5α和Rosetta-gami B（DE3）菌株中成功克隆并表达了EccB的截短重组蛋白，并通过亲和柱将其纯化，得到了一种38.6 kDa的蛋白。EccB及其突变体的截短形式均未检测到三磷酸腺苷酶活性。总之，我们的研究揭示了（）的eccB5基因截短形式的成功克隆和蛋白表达。ESX-5系统中的EccB蛋白可能是参与VII型分泌系统转运机制的重要膜成分，这对生长和毒力至关重要。
