Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure.
We have recently described the identification, isolation, and characterization of a factor, termed stem cell factor (SCF), which acts on primitive hematopoietic progenitors of the marrow. A soluble form of the factor was isolated from the conditioned medium of a rat cell line (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201) and rat and human cDNAs have been cloned (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C.-H., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211). The cDNAs encode amino acids C-terminal to those found in the isolated natural form, including a putative transmembrane domain. This paper describes the structural characterization of soluble forms of recombinant human SCF purified from Escherichia coli (unglycosylated) and from Chinese hamster ovary (CHO) cells (glycosylated). Fluorescence emission spectra indicate that the single Trp residue is present in a hydrophobic environment. Circular dichroism and infrared spectroscopy indicate considerable secondary structure, including both alpha-helix and beta-sheet. Molecular weight determinations by sedimentation equilibrium show that the molecules are dimeric (noncovalently associated), and gel filtration analyses are consistent with this conclusion. The CHO cell-derived SCF is about 30% carbohydrate by weight, with both N-linked and O-linked sugar. The presence or absence of the carbohydrate does not influence the results of the various structural analyses.
Arakawa
T
T
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Yphantis
D A
DA
Lary
J W
JW
Narhi
L O
LO
Lu
H S
HS
Prestrelski
S J
SJ
Clogston
C L
CL
Zsebo
K M
KM
Mendiaz
E A
EA
Wypych
J
J
D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D016298:Hematopoietic Cell Growth Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D019089:Stem Cell Factor; D013234:Stem Cells / Q000378:metabolism*
D016298:Hematopoietic Cell Growth Factors; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D006026:Glycoside Hydrolases