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Purification and partial characterization of recombinant human differentiation-stimulating factor.

C H Schmelzer·L E Burton·C M Tamony
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Abstract

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to greater than 95% purity from Chinese hamster ovary cells. RhD-factor is a glycoprotein with an apparent molecular weight of 45.6 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration in 6 M guanidine-hydrochloride, rhD-factor elutes with an apparent molecular weight of 21.5 kDa; it elutes with an apparent molecular weight of 44.8 kDa under neutral pH (native) conditions. The amino-terminal sequence (12 residues) is consistent with the expected sequence derived from the genomic DNA sequence. Recombinant D-factor is heavily glycosylated with 30% by weight neutral sugar and 12% sialic acid. The ED50 for rhD-factor was 0.25 ng/ml. Trifluoromethanesulfonic acid-deglycosylated rhD-factor has a biological activity comparable to that of the native recombinant protein (ED50 = 0.40 ng/ml). The biological activity of rhD-factor was stable at pH 1 for 40 h, in 6 M guanidine-HCl containing buffers with or without reducing agent, and in 1% SDS. Carboxymethylation of D-factor after reduction totally destroyed biological activity.

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Amino Acid SequenceAmino AcidsAnimalsCHO CellsChromatographyCricetinaeGlycosylationGrowth InhibitorsHumansHydrogen-Ion ConcentrationInterleukin-6Leukemia Inhibitory FactorLymphokinesMolecular Sequence DataMolecular WeightRecombinant ProteinsJournal Article

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C H Schmelzer, L E Burton, C M Tamony. 1990. Purification and partial characterization of recombinant human differentiation-stimulating factor.. https://doi.org/10.1016/1046-5928(90)90046-2

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