A simplified method for isolating outer membrane proteins from Pasteurella haemolytica A1

The outer membrane of Pasteurella haemolytica A1 (Ph-1) contains several major proteins which may play an important role in stimulating protective immunity. This report describes a method which was used to successfully isolate three Ph-1 outer membrane proteins (OMPs). The molecular weights of these OMPs are 31 kilodaltons (kD), 40 kD and 42 kD respectively. This method involves two steps: first, a sarcosyl extraction step was used to obtain a crude OMP prep; and secondly, these OMPs were separated by gel electrophoresis using a Bio-Rad 491 Prep Cell which separates proteins based on their molecular weights. Isolation of the proteins was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogenicity was then demonstrated by immunoblotting and enzyme-linked immuno-sorbent assays (ELISAs) with convalescent serum taken from calves with prior exposure to Ph-1. SDS-PAGE results show that single bands of each protein were obtained. Immunoblots and ELISAs of these proteins show that the convalescent antiserum used reacted with the 31 kD and 40 kD proteins, but did not react with the 42 kD protein. This procedure provides for a simplified method to obtain highly purified OMPs from Ph-1 which may now be evaluated on an individual basis as to what role they may play in stimulating resistance to pasteurellosis.