PURIFICATION AND CHARACTERIZATION OF A 31-KILODALTON IRON-REGULATED PERIPLASMIC PROTEIN FROM PASTEURELLA-HAEMOLYTICA A1

A prominent iron-regulated periplasmic protein was purified from Paste urella haemolytica grown in an iron-deficient chemically defined mediu m. The protein was purified by anion exchange chromatography and appea red 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 ave rage molecular weight of 29,877 as determined by analytical ultracentr ifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the prote in is referred to as the 31 kDa protein. Isoelectric focusing showed f our 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, EPFKVVTTFTVIQDIAQNV AGDKAT, showed a 95% identity with the 31 kDa iron-binding protein fro m Haemophilus influenzae. Isolation and characterization of iron-regul ated proteins are of particular interest because of their potential ro les in iron assimilation and microbial virulence.