Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography.
Stem cell factor (SCF) isolated from culture medium conditioned by Buffalo rat liver cells was subjected to detailed structural analysis. Attempts at direct N-terminal sequencing of the factor indicated that its N terminus is blocked as pyroglutamic acid (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., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201). The removal of the blocking pyroglutamate by pyroglutamate aminopeptidase allowed sequencing of the polypeptide chain to position 47. Stem cell factor was also digested with CNBr, trypsin, Staphylococcus aureus protease (strain V8), and AspN peptidase to generate different sets of peptides that were then separated by reverse-phase high-performance liquid chromatography and sequenced. Sequence of an internal peptide fragment obtained by cleavage of stem cell factor at a single tryptophanyl peptide bond was also obtained. From these analyses, the complete amino acid sequence could be constructed. The factor as isolated is a single polypeptide of 164 or 165 amino acids. The sequence is confirmatory to a sequence deduced from a cDNA sequence and provides important evidence for C-terminal processing of the polypeptide encoded by cDNA. There are four potential N-linked glycosylation sites. Asn65, Asn72, Asn109, and Asn120. Sequence determination of isolated peptides suggested that Asn120 is glycosylated, Asn65 and Asn109 glycosylated in some molecules but not in others, and Asn72 not glycosylated. Amino acids at three positions, i.e. 142, 143, and 155, could not be detected during sequence analysis. Since the gene sequence codes for Ser, Thr, and Thr at these positions (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. C., 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. W., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211), they could be sites of O-linked carbohydrate attachment. The four cysteines form two intramolecular disulfide bonds, Cys4-Cys89 and Cys43-Cys138.