Purification and characterization of a 31-kilodalton iron-regulated periplasmic protein from Pasteurella haemolytica A1.
A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9. The secondary structure of the protein was determined by circular dichroism and contained 16% alpha-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.
Tabatabai
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LB
National Animal Disease Center, USDA, ARS, Ames, Iowa 50010, USA.
Frank
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GH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000378:metabolism; D033862:Iron-Binding Proteins; D007525:Isoelectric Focusing; D016978:Mannheimia haemolytica / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D033863:Transferrin-Binding Proteins; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D033863:Transferrin-Binding Proteins; D007501:Iron