State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Jun;87(2):609-16. doi: 10.1007/s00253-010-2495-5. Epub 2010 Mar 6.
The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28 degrees C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.
炭疽毒素是一种 AB 型细菌毒素，由保护性抗原 (PA) 作为细胞结合的 B 组分，以及致死因子 (LF) 和水肿毒素 (EF) 作为催化的 A 组分组成。PA 组分是炭疽相关研究中的关键因素，重组 PA 通常可以在大肠杆菌中产生。然而，这种重组 PA 总是在大肠杆菌的细胞质中形成包涵体，使其纯化过程变得困难。在本研究中，我们发现通过与谷胱甘肽 S-转移酶 (GST) 融合并在 28°C 下诱导其表达，可以显著提高重组 PA 的可溶性。通过亲和层析法，从 1 升培养物中可获得 3 毫克蛋白质，从而实现了 PA 的高纯度纯化。此外，我们还表达和纯化了三种之前研究中表达受损的 PA 突变体，I394C、A396C 和 N435C。其中，鉴定出一种新型突变体 N435C，其对 PA 具有显性负抑制活性。这种新的突变体可能有助于设计用于炭疽预防和治疗的新型抗毒素。
