Abstract
A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.
Highlights
Million-fold by sequential fractionation using salting 30,000, and amino-terminalsequence analysis [14, 15]of each oucthromatographyc,hromatography on phenyl- protein gave sequences corresponding to partof the polypep
Regulatory factors for the proliferation and differentiation WEHI-3B cells may not be the same as the major species of of multipotential, erythroid, granulocyte-macrophage, eosin- multi-CSF produced by normal T-lymphocytes despite their ophil, megakaryocyte, and mastcell progenitor cells have been described from two main sources, medium conditioned by Tlymphocyte-related sources such as mitogen-stimulated Tlymphocytes ( l ), T-cell clones [2, 3], or T-hybridomas[4], and medium conditioned by the WEHI-3B myelomonocytic leukemia cell line [5,6].These sources have been reported to contain factors able to regulate the commitment of stem cells [7], the differentiation of late erythroid precursors [8] and the maintenance and proliferation of certain factor-dependent cell lines [9, 10]
Many of these activities have been sequence similarity [16, 17]. The resolution of this question requires the purification of the multi-CSF produced by Tlymphocytes
Summary
Purificationstudiesonother colony-stimulating factors have shown that as the protein concentration of the sample falls to levels below 50 pg/ml, losses of activity occur as a result of nonspecific adsorption of the proteins to plastic and glass surfaces [23,24,25]. Phenyl-Sepharose Chromatography-Thpeooled active fractions from the salting out stepwere in abuffer containing approximately 2.0 M ammonium sulfatein 0.1 M sodium phosphate buffer, pH 6.0 The presence of this high concentration of non-chaotropic saltin the sample allowed the pooled fractions to be loaded directly onto the phenyl-Sepharose column without further processing. All of the biological activities monitored co-eluted from the phenyl-Sepharose column at ammonium sulfate concentrations between 0.6 and 1.2 M (Fig. 2). This stepgave a good purification with excellent recovery and lowered the protein load of each batch to less than 5% of the initial concentrate.
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