Purification of a fibroblast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

A factor showing inhibitory activity against human gingival fibroblasts was extracted from the cytosol fraction of Actinobacillus actinomycetemocimitans Y4. The activity markedly inhibited the proliferation of human gingival fibroblasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemocomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.