Purification and characterization of the Pasteurella haemolytica 35 kilodalton periplasmic iron-regulated protein

Pasteurella haemolytica serovar Al is the causative agent of acute fibrinoh emorrahgic pneumonia also known as shipping fever. Many pathogens, includin g P. haemolytica, survive in their respective hosts through the up-regulati on of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 muM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA. The protein was purified to homogeneity by using ammonium sulfate precipita tion followed by CM-Sepharose ion exchange chromatography. SDS-PAGE showed a single band with a molecular weight of 35,369. Isoelectric focusing showe d multiple bands with pIs of 5.5, 5.6, 5.8, and one major band with pi of 6 .4. The molecular weight obtained by electrospray mass spectrometry was 35, 822. Equilibrium velocity ultracentrifugation established that the protein exist ed as a monomer under native conditions with an apparent molecular weight o f 33,795. Analysis of secondary structure of FbpA by circular dichroism sho wed 42.1% alpha helical structure. This protein is the second periplasmic i ron-regulated protein described for P. haemolytica.