Efficient Expression and Purification of Vacuolating Cytotoxin A (VacA) from an s1m2-type Thai Clinical Isolate
نویسندگان
چکیده
Vacuolating cytotoxin A, VacA, is one of the major virulence factors produced by H. pylori and its gene polymorphism might play a major role in strain virulence of H. pylori itself. VacA is produced as a 140 kDa protoxin which undergoes multiple cleavage reactions to give an 88 kDa mature toxin. The 88 kDa protoxin is further cleaved by unknown proteases into 55 kDa C terminal domain (p55) and 33 kDa N terminal domain (p33). The crystal structure of p55 was determined in 2007 by Gangwer et al, while p33 structure is yet to be solved. Several studies focusing on p33 reported that most of the expressed proteins are in insoluble form. Earlier work in our laboratory has demonstrated that expression of the pore-forming p33 domain in recombinant E. coli results in marked effects on cell growth which is stalled upon induction of protein biosynthesis. The scope of this project is to outline the strategy for optimum expression of recombinant s1m2 strain and to lay a ground work for the study of p33 domain, which crystal structure is yet to be solved. Here, we report the enhanced protein expression of Thai isolate VacA (s1m2 strain) by placing a DHFR fusion protein at the Nterminus of protein and efficient purification strategy for functional studies.
منابع مشابه
Sequence and Apoptotic Activity of VacA Cytotoxin Cloned from a Helicobacter pylori Thai Clinical Isolate
The vacuolating cytotoxin VacA produced by Helicobacter pylori induces the formation of large cytoplasmic vacuoles in host gastric epithelial cells as well as a release of cytochrome C from mitochondria resulting in cell apoptosis. Considerable sequence diversity in VacA relating to different degrees of disease severity is observed with clinical samples from a multitude of geographic places. In...
متن کاملExpression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Objective(s): Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. Materials and Methods: The antigenic region of V...
متن کاملAnalysis of expression of CagA and VacA virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided into two major types and that CagA is not necessary for expression of the vacuolating cytotoxin.
Colonization of the mucosa of the stomach and the duodenum by Helicobacter pylori is the major cause of acute and chronic gastroduodenal pathologies in humans. Duodenal ulcer formation strongly correlates with the expression of an antigen (CagA) that is usually coeexpressed with the vacuolating cytotoxin (VacA), a protein that causes ulceration in the stomach of mice. However, the relationship ...
متن کاملVacuolating Cytotoxin of Helicobacter pylori
Vacuolating cytotoxin (VacA) is one of the most important virulence factors of H. pylori (Hp), which isthe only toxic protein that is secreted from Hp cell into the culture supernatant. The effects of VacA oneukaryotic systems is the subject of many previous and on going research studies. Intracellular targetsfor this toxin include: late endosomal and lysosomal compartments, m...
متن کامل