title: Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa.
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.
authors.affiliation: Department of Microbiology and Immunology, Institute of Biology, The State University of Campinas, CP 6010, Campinas, SP, Brazil. cleidec@yahoo.com
authors.forename: Cleide Ferreira
authors.initials: CF
authors.lastname: Catani
authors.forename: Adriano Rodrigues
authors.initials: AR
authors.lastname: Azzoni
authors.forename: Débora Pires
authors.initials: DP
authors.lastname: Paula
authors.forename: Susely Ferraz Siqueira
authors.initials: SF
authors.lastname: Tada
authors.forename: Luciana Kauer
authors.initials: LK
authors.lastname: Rosselli
authors.forename: Anete Pereira
authors.initials: AP
authors.lastname: de Souza
authors.forename: Tomomasa
authors.initials: T
authors.lastname: Yano
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods*; D002845:Chromatography; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D044167:Xylella / Q000378:metabolism* / Q000472:pathogenicity*
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C077520:VapD protein, Bacteria; D007544:Isopropyl Thiogalactoside