tailieunhanh - Báo cáo khoa học: Expression of Helicobacter pylori CagA domains by library-based construct screening

Highly pathogenic strains ofHelicobacter pyloriuse a type IV secretion sys-tem to inject the CagA protein into human gastric cells. There, CagA asso-ciates with the inner side of the membrane and is tyrosine-phosphorylated at EPIYA motifs by host kinases. | Expression of Helicobacter pylori CagA domains by library-based construct screening Alessandro Angelini1 2 z Tommaso Tosi1 z Philippe Mas3 Samira Acajjaoui1 Giuseppe Zanotti2 Laurent Terradot1 and Darren J. Hart3 1 ESRF Grenoble France 2 Department of Chemistry Institute of Biomolecular Chemistry CNR University of Padua Italy 3 European Molecular Biology Laboratory Grenoble Outstation France Keywords CagA Helicobacter pylori protein expression type IV secretion system virulence factor Correspondence D. J. Hart European Molecular Biology Laboratory Grenoble Outstation 6 rue Jules Horowitz BP181 38042 Grenoble Cedex 9 France Fax 33 476 20 71 99 Tel 33 476 20 77 68 E-mail hart@ L. Terradot ESRF 6 Rue Jules Horowitz BP220 38043 Grenoble Cedex 9 France Fax 33 476 20 94 00 Tel 33 476 20 94 54 E-mail terradot@ These authors contributed equally to this work Received 22 October 2008 revised 1 December 2008 accepted 3 December 2008 doi Highly pathogenic strains of Helicobacter pylori use a type IV secretion system to inject the CagA protein into human gastric cells. There CagA associates with the inner side of the membrane and is tyrosine-phosphorylated at EPIYA motifs by host kinases. The phosphorylation triggers a series of interactions between CagA and human proteins that result in a dramatic change of cellular morphology. Structural and functional analyses of the protein have proved difficult due to the proteolytically sensitive nature of the recombinant protein. To circumvent these difficulties we applied ESPRIT a library-based construct screening method to generate a comprehensive set of 5 -randomly deleted gene fragments. Screening of 18 432 constructs for soluble expression resulted in a panel of 40 clones which were further investigated by large-scale purification. Two constructs of approximately 25 and 33 kDa were particularly soluble and were purified to near homogeneity. CagA fragments larger than 40 kDa were prone .