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J. Biol. Chem., Vol. 281, Issue 33, 27, August 18, 2006
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Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis
(Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102–1106)
Don W. Cleveland received his bachelor's degree in physics from New Mexico State University in 1972 and then enrolled in the graduate program in biochemistry at Princeton University. While in graduate school he developed a rapid method for the identification and characterization of proteins that exploited partial enzymatic proteolysis and analysis of the cleavage products by gel electrophoresis. This is the subject of the Journal of Biological Chemistry (JBC) Classic reprinted here.
Because of its speed, resolving power, adaptability, and ease of use, polyacrylamide gel electrophoresis had come into wide use after it was popularized by Klaus Weber and Mary Osborne in 1969. Weber and Osborne's seminal paper on using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the molecular weight of proteins appeared in the JBC and was the subject of a previous JBC Classic (1). The method was further improved by Ulrich K. Laemmli in 1970 when he pioneered the use of discontinuous gel electrophoresis (2). These gels, with stacking and separating layers, resulted in much higher resolution for mixtures of proteins.
Sometimes, however, the unambiguous identification of relationships between specific proteins cannot be made on the basis of electrophoretic mobility alone. This led to Cleveland's effort to produce a characteristic "peptide map" of proteolytic fragments from a purified polypeptide that could then be resolved using gel electrophoresis. Because small peptides are hard to resolve, Cleveland also needed to come up with digestion conditions that would produce large, stable fragments. He achieved this unexpectedly when he discovered that most proteases yielded large digestion products in the presence of sodium dodecyl sulfate.
Teaming up with Laemmli who was at Princeton at that time, Cleveland, along with Stuart G. Fischer and Marc W. Kirschner, used common proteases such as chymotrypsin and papain to partially digest several proteins. They found that the pattern of peptide fragments produced was characteristic of the protein substrate and the proteolytic enzyme and was highly reproducible. They also showed that a protein band in a gel slice could be analyzed without prior elution by its direct application to a resolving gel, which was then overlayed with a proteolytic enzyme. Thus, a very useful, rapid analytic technique requiring small amounts of protein was born.
"At that point," Cleveland recalls, "only the problem of publication remained. A manuscript was submitted to the Journal of Biological Chemistry, but to our amazement this was rejected without review by an associate editor who inferred erroneously that a more comprehensive paper was to be sent elsewhere. Happily, when we inquired, we encountered the unusual situation of having the same editor instruct us that a resubmitted manuscript without revision would be accepted forthwith (3)." The paper went on to become a Citation Classic, and as of 1984, it had been cited in over 1,830 publications. It is the 15th most cited article published in the JBC.
Cleveland received his Ph.D. from Princeton in 1977 and then moved to San Francisco where he became a postdoctoral fellow at the University of California, San Francisco, working with William Rutter. He later joined the faculty at Johns Hopkins University School of Medicine where he was an Assistant Professor (1981–1985), Associate Professor (1985–1988), and Professor (1988–1994) in the Department of Biological Chemistry. In 1995, Cleveland joined the University of California, San Diego (UCSD), School of Medicine.
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After publishing the JBC Classic with Cleveland, Laemmli joined the Department of Molecular Biology at the University of Geneva. He remains there today, researching chromosome structure and function.
One of Cleveland's coauthors on the paper, Marc W. Kirschner, has also established himself in the biological sciences. At the time the paper was published, Kirschner was an Assistant Professor at Princeton. In 1978 he became Professor at the University of California, San Francisco. In 1993, he moved to Harvard Medical School where he was founding chair of the Department of Systems Biology. Kirschner was elected a Foreign Member of the Royal Society of London and a Foreign Member of the Academia Europaea in 1999. He was the recipient of the 2001 ASBMB William C. Rose Award and a 2001 International Award by the Gairdner Foundation. Kirschner is a member of the National Academy of Sciences and the American Academy of Arts and Sciences. His laboratory at Harvard currently investigates the regulation of the cell cycle, the role of cytoskeleton in cell morphogenesis, and mechanisms of establishing the basic vertebrate body plan.
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