Construction and use of a prokaryotic expression system for Helicobacter pylori AhpC.
BACKGROUND
Helicobacter pylori is an important pathogen responsible for human gastric problems like inflammation, ulcers and cancer. It is widely prevalent in developing countries with low socioeconomic status. Since the infection remains asymptomatic in most individuals, efforts for efficient diagnostic markers to identify high risk patients are warranted. In this study, we constructed an expression vector that overexpresses the H. pylori AhpC protein as a glutathione S-transferase fusion protein. We furthermore examined whether this recombinant fusion protein retained immunogenicity and thus would be useful as a diagnostic marker.


RESULTS
The full-length tsaA gene from H. pylori strain G27, which encodes AhpC, was cloned in plasmid vector pGEX-6P-2 to create the recombinant plasmid vector pGEX-tsaA. The nucleotide sequence of the clone showed 100% homology with corresponding published sequence of original gene. Over-expression of the target protein GST-AhpC was achieved in E. coli BL21 (DE3) cells by induction with isopropyl-beta-D-thiogalactoside (IPTG). GST-AhpC was extracted and identified using SDS-PAGE as a 52 kDa protein. Western blotting results using commercial antibodies against whole cell H. pylori showed that the fusion protein retained immunogenecity.


CONCLUSIONS
A recombinant prokaryotic expression system was successfully established with high expression efficiency for target fusion gene pGEX-tsaA. The expressed GST-AhpC protein showed immunoreactivity against commercial anti-H. pylori antibodies. This recombinant fusion protein can be developed as a diagnostic marker for screening patients with chronic H. pylori infections.
Department of Microbiology, Quaid-i-Azam University, Islamabad, 45320, Pakistan. khalidadc@yahoo.co.uk
Khalid
K
Mehmood
Fariha
F
Hasan
D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis; D008970:Molecular Weight; D010544:Peroxidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D010544:Peroxidases; C508531:ahpC protein, Helicobacter pylori; D005982:Glutathione Transferase