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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.
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
February 1989,
Biochemistry,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
May 1992,
Applied biochemistry and biotechnology,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
April 1997,
Biotechnology and applied biochemistry,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
August 1991,
Biochemical and biophysical research communications,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
November 1989,
Biochemistry,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
January 1990,
Annals of the New York Academy of Sciences,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
June 1994,
Science in China. Series B, Chemistry, life sciences & earth sciences,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
May 2003,
Biochemical and biophysical research communications,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
April 1992,
Archives of biochemistry and biophysics,
C J Vlahos, and
O G Wilhelm, and
T Hassell, and
S R Jaskunas, and
N U Bang
July 2004,
Protein expression and purification,
