Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells.
Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.
Langley
K E
KE
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Wypych
J
J
Mendiaz
E A
EA
Clogston
C L
CL
Parker
V P
VP
Farrar
D H
DH
Brothers
M O
MO
Satygal
V N
VN
Leslie
I
I
Birkett
N C
NC
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D016298:Hematopoietic Cell Growth Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008407:Mast Cells / Q000187:drug effects; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019089:Stem Cell Factor
D016298:Hematopoietic Cell Growth Factors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019089:Stem Cell Factor