Protein Isolation and Separation Techniques of Pasteurella multocidavia One- and Two-Dimen-Sional Gel Electrophoresis

Abstract

Introduction: Bacteria in the genus Pasteurella live as commensal parasites on the mucous membranes of vertebrates, particularly mammals and birds. Pasteurella species like Pasteurella multocida and Pasteurella haemolytica cause significant economic losses as a result of diseases caused by these two microorganisms. Objective: This study compares protein isolation and analysis methods for effective one and two-dimensional gel electrophoresis of Pasteurella multocidaproteins and a preliminary protein mapping and analysis of P. multocida serotype B. Methodology: Protein samples were obtained by two isolation methods: homogenisation/sonication and detergent lysis. 1D SDS-PAGE methodology was optimized for protein loading, separating gel percentages and gel size. Detergent lysis was the preferred isolation method of total proteins. Results: The optimum running condition for SDS-PAGE was determined to be 15 µg of protein loading run on a 12.5% separating gel with a gel size of 10cm x 10cm. Preliminary 1D SDS-PAGE analysis revealed 3 distinct protein bands (33 kDa, 39 kDa and >200 kDa), which could only be found specifically in serotype B samples. The samples were then separated by 2-DGE after optimization. The optimum running condition was 500µg of protein sample loaded using rehydration loading with immobilized pH gradient (IPG) strips pH 3–10 for the first-dimension isoelectric focusing (IEF) and 12.5% single percentage gel for the seconddimension SDS-PAGE. The protein map, which produced the most, spots were compared to in silico 2-DGE protein patterns of Pm70 in GELBANK database. Conclusion: A total of 23 proteins were identified and protein of 33 kDA was identified in both 1D SDS-PAGE and 2-DGE. This is the first protein analysis and the first protein map of P. multocida serotype B ever produced.