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Towards large‐scale purification of natural CSF‐1 from human placenta tissue extracts
Author(s) -
MartinThouvenin V.,
Uhlrich S.,
Tayot JL,
Lanotte M.
Publication year - 1990
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1990.tb00090.x
Subject(s) - chromatography , isoelectric point , chemistry , biological activity , high performance liquid chromatography , size exclusion chromatography , gel permeation chromatography , affinity chromatography , chromatofocusing , biochemistry , in vitro , enzyme , organic chemistry , polymer
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.