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Purification and characterization of a 31-kilodalton iron-regulated periplasmic protein from Pasteurella haemolytica A1

Tabatabai, L.B., Frank, G.H.
Preparative biochemistry & biotechnology 1997 v.27 no.4 pp. 253-269
Haemophilus influenzae, Mannheimia haemolytica, anion exchange chromatography, circular dichroism spectroscopy, desorption, iron, isoelectric focusing, mass spectrometry, molecular weight, nutrient deficiencies, polyacrylamide gel electrophoresis, protein secondary structure, proteins, ultracentrifugation, virulence
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.