Abstract
In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37 degrees C, with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Bacterial Proteins / chemistry
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Bacterial Proteins / physiology*
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Base Sequence
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Biochemistry / methods*
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Chromatography
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Circular Dichroism
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Cloning, Molecular
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / metabolism
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Genetic Vectors
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Isopropyl Thiogalactoside / chemistry
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Mass Spectrometry
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Membrane Glycoproteins / chemistry
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Membrane Glycoproteins / physiology*
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Molecular Sequence Data
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Open Reading Frames
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Plasmids / metabolism
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Polymerase Chain Reaction
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / chemistry
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Sequence Homology, Nucleic Acid
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Time Factors
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Xylella / metabolism*
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Xylella / pathogenicity*
Substances
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Bacterial Proteins
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Membrane Glycoproteins
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Recombinant Fusion Proteins
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VapD protein, Bacteria
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Isopropyl Thiogalactoside