Overexpression and purification of Helicobacter pylori flavodoxin and induction of a specific antiserum in rabbits.
Flavodoxin from the gastric pathogen Helicobacter pylori has been shown to be the electron acceptor of the essential pyruvate-oxidoreductase enzyme complex and proposed to be involved in the pathogenesis of gastric MALToma. In order to obtain a sufficient amount for biochemical and structural studies, we overexpressed the protein either with a C-terminal His(6) -tag or as a fusion protein upstream of intein- and chitin-binding domains. With both expression systems we succeeded at purifying soluble and functional flavodoxin containing the cofactor FMN. When expressing with a His(6) -tag, we purified approximately 20 mg flavodoxin per liter of bacterial culture, while expression as an intein-CBD fusion protein with autocatalytic removal of the intein-CBD part rendered only approximately 1 mg of purified flavodoxin per liter of bacterial culture. Expressed as an intein-CBD fusion protein, flavodoxin copurified with a C-terminal degradation product, which was not observed for expression with a His(6) -tag. However, we were able to obtain protein crystals suited for X-ray structure determination from flavodoxin expressed as an intein-CBD fusion protein, but not from flavodoxin expressed with a C-terminal His(6) -tag. We further report the induction of a rabbit antiserum specific for H. pylori flavodoxin.
Paul
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Division of Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland. Ralf.Paul@Yale.edu
Bosch
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Schäfer
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D000818:Animals; D003001:Cloning, Molecular; D005418:Flavodoxin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D007106:Immune Sera / Q000096:biosynthesis; D011817:Rabbits / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D005418:Flavodoxin; D007106:Immune Sera; D011993:Recombinant Fusion Proteins