Reversible self-association of recombinant bovine factor B

Grigory I. Belogrudov, Virgil Schirf, Borries Demeler

Research output: Contribution to journalArticlepeer-review

Abstract

The recombinant bovine factor B, obtained by a newly developed bacterial expression system, was found to exhibit features characteristic of a reversible self-associating system. Using size-sieving chromatography, distribution of the factor B species ranged from a monomer to a trimer, but not oligomers of higher molecular weights. At high protein concentrations, factor B migrated as a single band in a native gel. Cross-linking with the amino-reactive cross-linking reagent bis (sulfosuccinimidyl) suberate (BS), at a low cross-linker to protein ratio yielded cross-linked products identified as factor B dimer and trimer. The cross-linking pattern was shown to be a function of the protein and cross-linker concentrations. The range of sedimentation coefficients in a sedimentation velocity experiment suggested that the largest particle present in the distribution was more than twice as large as the smallest. The data obtained under multiple conditions in the sedimentation equilibrium experiments are best fit to a model describing a reversible self-association of a monomer-trimer of factor B species, with a dissociation constant Kd1,3 = 2.48 × 10- 10 M2.

Original languageEnglish
Pages (from-to)1741-1749
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1764
Issue number11
DOIs
StatePublished - Nov 2006

Keywords

  • Analytical ultracentrifugation
  • Cross-link
  • Dimer
  • Energy coupling
  • Factor B
  • Proton leak
  • Sedimentation equilibrium
  • Sedimentation velocity
  • Self-association
  • Trimer
  • UltraScan

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