Expression of Helicobacter pylori CagA domains by library-based construct screening.
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 to heavy proteolysis at the C-terminus, with a favoured cleavage site near the first EPIYA motif. Thus, these well-expressed recombinant constructs isolated are likely to be similar to those observed following natural proteolysis in human cells, and open the way for structural and functional studies requiring large amounts of purified material.
Angelini
Alessandro
A
ESRF, Grenoble, France.
Tosi
Tommaso
T
Mas
Philippe
P
Acajjaoui
Samira
S
Zanotti
Giuseppe
G
Terradot
Laurent
L
Hart
Darren J
DJ
D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D030561:Databases, Nucleic Acid; D017353:Gene Deletion; D015870:Gene Expression; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D010446:Peptide Fragments; C080866:cagA protein, Helicobacter pylori