Summary
Peptide fragments have been widely used in biophysical studies on specific regions of integral membrane proteins. Because of their inherent insoluble nature and tendency to aggregate the preparation of such model peptides is challenging. We have developed synthetic and biosynthetic approaches to prepare peptides containing single and multiple domains of a G protein–coupled receptor. Both the synthetic and biosynthetic products can be isolated by reversed-phase highperformance liquid chromatography to near homogeneity. The biosynthetic product, a fusion protein, is processed by CNBr cleavage to yield the target peptide in various isotopic forms. The final peptides are studied by circular dichroism spectroscopy to determine their secondary structure under a variety of conditions.
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Acknowledgments
Many talented and dedicated students have participated in the development of the methods outlined in this chapter. They are: Enrique Arevalo, Boris Arshava, Michael Breslav, Patricia Cano-Sanchez, Fa-Xiang Ding, Gary Eng, Jacqueline Englander, Racha Estephan, Sanjay Khare, Shufing Liu, Jennifer Madeo, Abdulla Reddy, Joseph Russo, David Schreiber, Beatrice Severino, V.V. Sureshbabu, and Haibo Xie. I am indebted to all of them. I also owe much to my lifelong friend and collaborator Professor Jeffrey M. Becker who introduced me to Saccharomyces cerevisiae and who has stimulated my laboratory to conduct analyses of G protein–coupled receptors. I also wish to thank Racha Estephan, Jacqueline Englander, Leah Cohen, Boris Arshava, and Jeff Becker for carefully reading this chapter and for their helpful suggestions. All of these studies have been supported from by a grant from the National Institute of General Medical Sciences (GM 22086).
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Naider, F. (2007). Synthesis, Biosynthesis, and Characterization of Transmembrane Domains of a G Protein—Coupled Receptor. In: Fields, G.B. (eds) Peptide Characterization and Application Protocols. Methods in Molecular Biology™, vol 386. Humana Press. https://doi.org/10.1007/978-1-59745-430-8_4
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DOI: https://doi.org/10.1007/978-1-59745-430-8_4
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