期刊
PROTEIN AND PEPTIDE LETTERS
卷 28, 期 6, 页码 643-650出版社
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/0929866527666201112122831
关键词
Cell viability assay; His((6)) tag; human gastric cell; hydrophobic stretch; inclusion solubilization; membrane perturbation
资金
- Thailand Research Fund (TRF) [RSA5580047]
The study found that the N-terminal tag played a crucial role in enhancing the expression of Helicobacter pylori VacA variants. Full-length VacA-m1 and the 33-kDa domain required N-terminal extension for efficient expression, while the 55-kDa domain could be expressed with either N or C-terminal extension. Despite higher membrane-perturbing and cytotoxic activities of VacA-m1 compared to VacA-m2, further research is needed to explore the differences between the domains.
Background: Gastric pathogen Helicobacter pylori secretes VacA cytotoxin displaying a high degree of polymorphic variations of which the highest VacA pathogenicity correlates with m1-type variant followed by VacA-m2. Objective: To comparatively evaluate expression in Escherichia coli of the mature VacA variants (m1- and m2-types) and their 33- and 55/59-kDa domains fused with His((6)) tag at N- or C-terminus. Methods: All VacA clones expressed in E. coli TOP10 (TM) were analyzed by SDS-PAGE and Western blotting. VacA inclusions were solubilized under native conditions (similar to 150-rpm shaking at 37 degrees C for 2 h in 20 mM HEPES (pH7.4) and 150 mM NaCl). Membrane-perturbing and cytotoxic activities of solubilized VacA proteins were assessed via liposome-entrapped dye leakage and resazurin-based cell viability assays, respectively. VacA binding to human gastric adenocarcinoma cells was assessed by immunofluorescence microscopy. Side-chain hydrophobicity of VacA was analyzed through modeled structures constructed by homology- and ab initio-based modeling. Results: Both full-length VacA-m1 and 33-kDa domain were efficiently expressed only in the presence of N-terminal extension while its 55-kDa domain was capably expressed with either N- or C-terminal extension. Selectively enhanced expression was also observed for VacA-m2. Protein expression profiles revealed a critical period in IPTG-induced production of the 55-kDa domain with N-terminal extension unlike its C-terminal extension showing relatively stable expression. Both VacA-m1 isolated domains were able to independently bind to cultured gastric cells similar to the full-length toxin, albeit the 33-kDa domain exhibited significantly higher activity of membrane perturbation than others. Membrane-perturbing and cytotoxic activities observed for VacA-m1 appeared to be higher than those of VacA-m2. Homology-based modeling and sequence analysis suggested a potential structural impact of non-polar residues located at the N-terminus of the mature VacA toxin and its 33-kDa domain. Conclusion: Our data provide molecular insights into selective influence of the N-terminally added tag on efficient expression of recombinant VacA variants, signifying biochemical and biological implications of the hydrophobic stretch within the N-terminal domain.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据