Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa.
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.
Catani
Cleide Ferreira
CF
Department of Microbiology and Immunology, Institute of Biology, The State University of Campinas, CP 6010, Campinas, SP, Brazil. cleidec@yahoo.com
Azzoni
Adriano Rodrigues
AR
Paula
Débora Pires
DP
Tada
Susely Ferraz Siqueira
SF
Rosselli
Luciana Kauer
LK
de Souza
Anete Pereira
AP
Yano
Tomomasa
T
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*
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C077520:VapD protein, Bacteria; D007544:Isopropyl Thiogalactoside