Detection and Rapid Purification of Internalin B as a Protein Marker in Listeria monocytogenes

Abstract

Clinical and food strains of Listeria monocytogenes have been found to express InternalinB (InlB) without polymorphism. InlB, which is a 67-kDa surface protein, behaves as an invasion and adhesion protein of the bacterium into cells. Thus, InlB could be a good candidate as a protein marker to detect L. monocytogenes. Three strains of L. monocytogenes (ATCC 19115, serotype 4b, ATCC 19111, serotype 1/2a, ATCC 7644, serotype 1/2c) were tested to detect and purify InlB. L. grayii (ATCC 25400) and L. innocua (isolated from soft cheese) were used as controls that did not express InlB due to the absence of its gene on the chromosome. InlB was quantitatively extracted in a solubilized form by treatment of L. monocytogenes with 1 M Tris-Cl at pH 7.5. Immunoblot analysis using anti-InlB polyclonal antibody revealed that L. monocytogenes 19115 had the lowest expression, which required enrichment of InlB for its detection. Simple spin-ion exchange chromatography with strong acidic cation exchanger was used to enrich the InlB protein that is strongly basic. Most impurities in the column were washed with 25 mM sodium acetate whereas the InlB protein was only protein retained in the column and can be eluted by 1 M NaCl. The data presented here showed that spin-ion exchange chromatography was found to be a simple and rapid method to enrich and purify InlB within 20 min.

Key Words:

• InlB
• Listeria monocytogenes
• 1 M Tris-Cl extraction
• rapid detection method
• Spin-ion exchange chromatography

ACKNOWLEDGMENTS

Approved for publication as Journal Article No. J-11003 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. This work was supported in part by the Mississippi Agricultural and Forestry Experiment Station Project Number MIS-371272 and by USDA-ARS Grant No. 58-0790-5-137. We gratefully acknowledge Dr. Arun K. Bhunia (Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University) for kindly providing anti-Listeria InlB antibody.