Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum

• Published in: Protein expression and purification 2005-03, Vol.40 (1), p.152-163
• Main Authors: Goodin, Jeremy L., Raab, Ronald W., McKown, Robert L., Coffman, George L., Powell, Bradford S., Enama, Jeff T., Ligon, John A., Andrews, Gerard P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - immunology; Bacterial Outer Membrane Proteins - isolation & purification; Base Sequence; Escherichia coli - genetics; His-tag; Immune Sera - biosynthesis; Intein; Membrane Transport Proteins - genetics; Membrane Transport Proteins - immunology; Membrane Transport Proteins - isolation & purification; Mice; Molecular Sequence Data; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - pharmacology; Type III secretion; Yersinia pestis; yscC
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.11.001

The type III secretion system (YscC) protein of Yersinia pestis plays an essential role in the translocation of Yersinia outer proteins (Yops) into eukaryotic target cells through a type III secretion mechanism. To assess the immunogenicity and potential protective efficacy of YscC against lethal plague challenge, we cloned, overexpressed, and purified YscC using two different bacterial expression and purification systems. The resulting expression plasmids for YscC, pETBlue-2-YscC and pTYB11-YscC, were regulated by robust T7 promoters that were induced with isopropyl-β- d-thiogalactopyranoside. The intein–fusion pTYB11-YscC system and the six-histidine-tagging pETBlue-2-YscC system were both successful for producing and purifying YscC. The intein-mediated purification system produced about 1 mg of soluble YscC per liter of bacterial culture while the YscC-His 6-tag method resulted in 16 mg of insoluble YscC per liter of bacterial culture. Protein identity for purified YscC-His 6 was confirmed by ion trap mass spectrometry. Antisera were produced against both YscC and YscC-His 6. The specific immune response generated in YscC-vaccinated mice was relative to the particular purified protein, YscC or YscC-His 6, which was used for vaccination as determined by Western blot analysis and ELISA. Regardless of the purification method, either form of the YscC protein failed to elicit a protective immune response against lethal plague challenge with either F1 capsule forming Y. pestis CO92 or the isogenic F1 − Y. pestis C12.