[Development and characterization of Helicobacter pylori CagA recombinant fragments].
OBJECTIVE
The goal of the work was to produce, purify and characterize recombinant fragments of Helicobacter pylori CagA protein.


METHODS
The methods of molecular cloning, recombinant protein expression in Escherichia coli, affinity chromatography, gel electrophoresis and western-blotting as well as several in silico algorithms of nucleotide and aminoacid sequence analysis were used.


RESULTS
Four N-terminal His6-tagged recombinant fragments of CagA protein were produced. Protein rCagAfr.1 (65 kDa) represents the most conserved N-terminal part of the cytotoxin. Fragment rCagAfr.2 (44 kDa) corresponds to the central conserved region of CagA whereas rCagAfr.3 (39 kDa) represents the highly variable C-terminal part of CagA. Finally, the protein rCagAfr.4 (75 kDa) incorporates the sequences of rCagAfr.2 and rCagAfr.3. In silico analysis of fragments' sequences allowed to suppose that rCagAfr.2 and rCagAfr.4 are highly immunogenic proteins. By means of chromatographic purification, high levels of purity (up to 97%) and yield (about 15 mg per L of culture) of recombinant proteins were achieved.


CONCLUSIONS
Use of recombinant proteins technology allowed to solve the problem of producing the CagA pure protein of H. pylori, which open new perspectives for the development of immunodiagnostic assays for detection of CagA protein or antibodies to this cytotoxin.
Klimovich
A V
AV
Samoĭlovich
M P
MP
Suvorov
A N
AN
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016481:Helicobacter Infections / Q000175:diagnosis*; D016480:Helicobacter pylori / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007159:Immunologic Tests; D010446:Peptide Fragments / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C080866:cagA protein, Helicobacter pylori