PROPERTIES OF VARIANT FORMS OF HUMAN STEM-CELL FACTOR RECOMBINANTLY EXPRESSED IN ESCHERICHIA-COLI

The gene for human stem cell factor (SCF) encodes a leader sequence fo llowed by 248 amino acids (Martin et al., 1990, Cell 63, 203). Of thes e 248 amino acids, the first 189 correspond to an extracellular domain and the remainder correspond to a hydrophobic transmembrane domain pl us a cytoplasmic domain. A naturally occurring soluble form, released by proteolytic cleavage after amino acid 165, has been described. An a lternatively spliced mRNA, lacking the codons for exon 6, has also bee n described. Since the amino acids encoded by exon 6 include the prote olytic cleavage site, the form expressed from the alternatively splice d mRNA tends to remain membrane-bound. In the present study, we have b egun to explore structure/function relationships within the extracellu lar domain of SCF. Forms beginning at amino acid 1 (after the leader s equence) and ranging from 127 to 189 at the C-terminus have been recom binantly expressed in Escherichia coli and purified. In addition, form s missing the amino acids encoded by exon 6, forms missing up to 10 am ino acids from the N-terminus, and forms with disulfide bond alteratio ns have been expressed and purified. The forms have been characterized structurally, as well as functionally, in quantitative cell prolifera tion and receptor-binding assays. The results indicate that amino acid s 1-141 comprise a structural and functional core and allow conclusion s about the necessity of each of the two disulfide bonds for structure and function. (C) 1994 Academic Press, Inc.