Overexpression and Purification of Helicobacter pylori Flavodoxin and Induction of a Specific Antiserum in Rabbits

Paul, Ralf; Bosch, Frank U; Schäfer, Klaus

doi:10.1006/prep.2001.1467

Cited by 12 publications

(8 citation statements)

References 37 publications

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“…Recombinant H. pylori flavodoxin was purified as described (Paul et al 2001). Shortly, the fld A gene of the H. pylori strain 69A was PCR‐amplified and cloned into the Nde I and Sap I restriction sites of the pTYB1 expression vector (New England Biolabs [NEB]).…”

Section: Methodsmentioning

confidence: 99%

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Crystal structure of oxidized flavodoxin, an essential protein in Helicobacter pylori

et al. 2002

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The redox protein flavodoxin has been shown earlier to be reduced by the pyruvate-oxidoreductase (POR) enzyme complex of Helicobacter pylori, and also was proposed to be involved in the pathogenesis of gastric mucosa-associated lymphoid-tissue lymphoma (MALToma). Here, we report its X-ray structure, which is similar to flavodoxins of other bacteria and cyanobacteria. However, H. pylori flavodoxin has an alanine residue near the isoalloxazine ring of its cofactor flavin mononucleotide (FMN), while the other previously crystallized flavodoxins have a larger hydrophobic residue at this position. This creates a solute filled hole near the FMN cofactor of H. pylori flavodoxin. We also show that flavodoxin is essential for the survival of H. pylori, and conclude that its structure can be used as a starting point for the modeling of an inhibitor for the interaction between the POR-enzyme complex and flavodoxin.

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Section: Methodsmentioning

confidence: 99%

“…For immunostaining, proteins were transferred onto nitrocellulose membranes (Protran BA85 Schleicher & Schüll; Towbin et al 1979). The antiserum against flavodoxin was used in a 1:25,000 dilution (Paul et al 2001). The secondary, antirabbit antibody (Diagen), which was conjugated to horseradish‐peroxidase, was detected by ECL (Amersham).…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of oxidized flavodoxin, an essential protein in Helicobacter pylori

et al. 2002

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“…This is of great interest since they include human pathogens such as Helicobacter pylori [39]. Flavodoxin is thus a potential target for drug design [39,[104][105][106]. Indeed, the flavodoxin from Helicobacter pylori displays a peculiar structural feature that can be exploited to bind inhibitory molecules next to the active site [9].…”

Section: Future Researchmentioning

confidence: 99%

Flavodoxins: sequence, folding, binding, function and beyond

Sancho

2006

Cell. Mol. Life Sci.

183

234

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Flavodoxins are electron-transfer proteins involved in a variety of photosynthetic and non-photosynthetic reactions in bacteria, whereas, in eukaryotes, a descendant of the flavodoxin gene helps build multidomain proteins. The redox activity of flavodoxin derives from its bound flavin mononucleotide cofactor (FMN), whose intrinsic properties are profoundly modified by the host apoprotein. This review covers the very exciting last decade of flavodoxin research, in which the folding pathway, the structure and stability of the apoprotein, the mechanism of FMN recognition, the interactions that stabilize the functional complex and tailor the redox potentials, and many details of the binding and electron transfer to partner proteins have been revealed. The next decade should witness an even deeper understanding of the flavodoxin molecule and a greater comprehension of its many physiological roles. The fact that flavodoxin is essential for the survival of some human pathogens could make it a drug target on its own.

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“…In one report, both N-and C-terminal intein fusions failed to be cleaved even though the amino acids adjacent to the cleavage sites are favorable ones, suggesting that the cleavage might be blocked by steric hindrance (Wu and Oppermann 2003). In addition to incomplete and failed processing, cleavage at unintended sites has also been reported (Paul et al 2001;Luka and Wagner 2003). Another drawback of the intein system is the uncontrolled in vivo cleavage, which in some cases could leave most of the fusion proteins cleaved during expression, thus causing poor yield of the target protein (Wood 2003;Cui et al 2006).…”

Section: Available Self-cleaving Fusion Tagsmentioning

confidence: 92%

Self-cleaving fusion tags for recombinant protein production

2011

Biotechnol Lett

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Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.

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Overexpression and Purification of Helicobacter pylori Flavodoxin and Induction of a Specific Antiserum in Rabbits

Cited by 12 publications

References 37 publications

Crystal structure of oxidized flavodoxin, an essential protein in Helicobacter pylori

Crystal structure of oxidized flavodoxin, an essential protein in Helicobacter pylori

Flavodoxins: sequence, folding, binding, function and beyond

Self-cleaving fusion tags for recombinant protein production

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