Purification and characterization of the Pasteurella haemolytica 35 kilodalton periplasmic iron-regulated protein.
Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrhagic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation 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 gM 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 precipitation followed by CM-Sepharose ion exchange chromatography. SDS-PAGE showed a single band with a molecular weight of 35,369. Isoelectric focusing showed 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 existed as a monomer under native conditions with an apparent molecular weight of 33,795. Analysis of secondary structure of FbpA by circular dichroism showed 42.1% alpha helical structure. This protein is the second periplasmic iron-regulated protein described for P. haemolytica.
Belzer
C A
CA
Center for Veterinary Biologics Licensing and Policy Development Ames, IA 50010, USA.
Tabatabai
L B
LB
Frank
G H
GH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron; D033862:Iron-Binding Proteins; D007525:Isoelectric Focusing; D016978:Mannheimia haemolytica / Q000737:chemistry*; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014461:Ultracentrifugation / Q000379:methods
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D033862:Iron-Binding Proteins; D033902:Periplasmic Binding Proteins; D007501:Iron