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Title: PURIFICATION AND CHARACTERIZATION OF THE PASTEURELLA HAEMOLYTICA 35- KILODALTON PERIPLASMIC IRON-REGULATED PROTEIN

Author
item BELZER, CAROL
item Tabatabai, Louisa
item Frank, Glynn

Submitted to: Preparative Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/15/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Pasteurella haemolytica serovar A1 is a Gram-negative bacterium and a bacterial pathogen that causes bovine respiratory disease (BRD) also known as shipping fever. We have purified a potential virulence factor, which is an iron-binding protein responsible for transport of iron and plays a role in the survival of the pathogen. Serum from calves taken three days after infection with Pasteurella haemolytica serovar A1 shows a weak immunoglobulin G reaction. Serum from calves that have recovered from infection show a very strong immunoglobulin G reaction with the iron transport protein. The results suggest that this protein will serve as a diagnostic antigen for shipping fever and the protein may also serve as a potential component for an improved vaccine.

Technical Abstract: A 35-kilodalton periplasmic iron-regulated protein from P. haemolytica serovar A1 has been isolated, purified and characterized. The protein was identified as the FbpA homologue of the H. influenzae iron transport protein. A synthetic medium without iron and supplemented with 50 uM 2,2' dipyridyl facilitated the expression, isolation and purification of the protein. The periplasmic proteins were precipitated from osmotic shock fluids with a two-step ammonium sulfate precipitation procedure. The FbpA was purified to homogeneity by CM-Sepharose ion exchange chromatography. Isoelectric focusing showed three minor bands with pIs of 5.5, 5.6, 5.8, and one major band with pI of 6.4. Mr of the FbpA ranged from 35,369 (SDS-PAGE) to 35,822 (ES-MS); other methods for molecular weight determination included MALDI-TOF (Mr of 35,980) and amino acid analysis (Mr of 34,810). Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with an Mr of 33,795. The N-terminal sequence (ANEVNVYSYRQPYLIEPMLK) and amino acid composition were determined from protein blotted to polyvinylidene fluoride (PVDF) microporous membrane. The database revealed that the N-terminal sequence of the 35-kDa periplasmic iron-regulated protein was identical to the FbpA homologue of H. influenzae. Although the FbpA reacted weakly with serum IgG from acutely infected animals, the protein may serve as a diagnostic protein for the detection of the infectious agent in respiratory disease and the protein may also serve as a potential component for an improved vaccine.