Genomic Cloning and Heterologous Expression of Human Differentiation-Stimulating Factor

Abstract

Differentiation-stimulating factor (D-factor) purified from mouse Ehrlich ascites cells was sequenced partially and found to be almost identical to leukemia inhibitory factor (LIF) from mouse Krebs II ascites cells. In comparison to LIF, D-factor had an additional amino-terminal serine residue. Using synthetic oligonucleotide probes designed from the murine D-factor sequence, we cloned the human gene encoding D-factor. A partial D-factor cDNA was cloned from COS-1 cells transfected with the human D-factor gene under the control of a heterologous promoter. We used this cDNA to construct a vector for direct expression of the protein in Escherichia coli. A mammalian cell expression vector was constructed using the signal sequence of interferon-αA linked to the D-factor cDNA. Both forms of recombinant human D-factor were active on the murine myeloid leukemia cell line M1 in a dose- and time-dependent manner for the inhibition of [³H]thymidine incorporation, and also induced phagocytosis, Fc receptor expression, and prostaglandin E₂ synthesis by M1 cells.