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N-Terminally Added Tag Selectively Enhances Heterologous Expression of VacA Cytotoxin Variants from Helicobacter pylori

Author(s):

Aung Khine Linn, Nitchakan Samainukul, Somsri Sakdee, Chonthicha Butnampetch, Hui-Chun Li, Chanan Angsuthanasombat* and Gerd Katzenmeier   Pages 1 - 9 ( 9 )

Abstract:


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-typtes) 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 were analyzed by SDS-PAGE and Western blotting. VacA inclusions were solubilized under native conditions (˜150-rpm shaking at 37°C for 2 h in 20 mM HEPES, pH 7.4 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 provides 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.

Keywords:

Cell viability assay, His(6) tag, human gastric cells, hydrophobic stretch, inclusion solubilization, membrane perturbation

Affiliation:

Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Department of Biochemistry, School of Medicine, Tzu Chi University, Hualian 97004, Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170



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