Towards large-scale purification of natural CSF-1 from human placenta tissue extracts.
The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.
Centre Hayem, Hôpital Saint-Louis, Paris, France.
V
V
Martin-Thouvenin
S
S
Uhlrich
J L
JL
Tayot
M
M
Lanotte
D015153:Blotting, Western; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003115:Colony-Stimulating Factors / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D016173:Macrophage Colony-Stimulating Factor; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy
D003115:Colony-Stimulating Factors; D008623:Mercaptoethanol; D016173:Macrophage Colony-Stimulating Factor