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 followed by 248 amino acids (Martin et al., 1990, Cell 63, 203). Of these 248 amino acids, the first 189 correspond to an extracellular domain and the remainder correspond to a hydrophobic transmembrane domain plus a cytoplasmic domain. A naturally occurring soluble form, released by proteolytic cleavage after amino acid 165, has been described. An alternatively spliced mRNA, lacking the codons for exon 6, has also been described. Since the amino acids encoded by exon 6 include the proteolytic cleavage site, the form expressed from the alternatively spliced mRNA tends to remain membrane-bound. In the present study, we have begun to explore structure/function relationships within the extracellular domain of SCF. Forms beginning at amino acid 1 (after the leader sequence) and ranging from 127 to 189 at the C-terminus have been recombinantly expressed in Escherichia coli and purified. In addition, forms missing the amino acids encoded by exon 6, forms missing up to 10 amino acids from the N-terminus, and forms with disulfide bond alterations have been expressed and purified. The forms have been characterized structurally, as well as functionally, in quantitative cell proliferation and receptor-binding assays. The results indicate that amino acids 1-141 comprise a structural and functional core and allow conclusions about the necessity of each of the two disulfide bonds for structure and function.
Langley
K E
KE
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Mendiaz
E A
EA
Liu
N
N
Narhi
L O
LO
Zeni
L
L
Parseghian
C M
CM
Clogston
C L
CL
Leslie
I
I
Pope
J A
JA
Lu
H S
HS
D017398:Alternative Splicing; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D016298:Hematopoietic Cell Growth Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D019089:Stem Cell Factor; D013329:Structure-Activity Relationship
D004220:Disulfides; D016298:Hematopoietic Cell Growth Factors; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019089:Stem Cell Factor