Disulfide pairing of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.
The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U. (1990) J. Biol. Chem. 265, 14606-14611), was internally radiolabeled using [35S]methionine-cysteine. Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography. The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography. Five major peptides containing 35S were obtained. Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al. (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G. A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D. (1983) Nature 301, 214-221)). These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies.
Vlahos
C J
CJ
Lilly Research Laboratories, Indianapolis, Indiana 46285.
Wilhelm
O G
OG
Hassell
T
T
Jaskunas
S R
SR
Bang
N U
NU
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013820:Thermolysin; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D013820:Thermolysin