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Updated: Aug 22, 2026
A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction
Published on: May 17, 2024
Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa
Cleide Ferreira Catani1, Adriano Rodrigues Azzoni, Débora Pires Paula
1Department of Microbiology and Immunology, Institute of Biology, The State University of Campinas, CP 6010, Campinas, SP, Brazil. cleidec@yahoo.com
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.
