Protein Engineering Protocols

Volume 352 of the series Methods in Molecular Biology™ pp 249-274

Synthesis of Degenerated Libraries of the Ras-Binding Domain of Raf and Rapid Selection of Fast-Folding and Stable Clones With the Dihydrofolate Reductase Protein Fragment Complementation Assay

  • François-Xavier Campbell-ValoisAffiliated withDépartement de Biochimie, Université de Montréal
  • , Stephen W. MichnickAffiliated withDépartement de Biochimie, Université de Montréal

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The protein-engineering field is mainly concerned with the design of novel enzyme activities or folds and with understanding the fundamental sequence determinants of protein folding and stability. Much effort has been put into the design of methods to generate and screen libraries of polypeptides. Screening for the ability of proteins to bind with high affinity and/or specificity is most often approached with phage display technologies. In this chapter, we present an alternative to phage display, performed totally in vivo, based on the dihydrofolate reductase (DHFR) protein-fragment complementation assay (PCA). We describe the application of the DHFR PCA to the selection of degenerated sequences of the ras-binding domain (RBD) of raf for correct folding and binding to ras. Our screening system allows for enrichment of the libraries for the best-behaving sequences through iterative competition experiments, without the discrete library screening and expansion steps that are necessary in in vitro approaches. Moreover, the selected clones can be processed rapidly to purification by Ni-nitrilotriacetic acid (NTA) affinity chromatography in 96-well plates. Our methods are particularly suitable for the designing and screening of libraries aimed at studying sequence folding and binding determinants. Finally, it can be adapted for library-against-library screening, thus, allowing for coevolution of interacting proteins simultaneously.

Key Words

Protein-fragment complementation assays dihydrofolate reductase bacterial survival assay phage display protein-protein interactions protein engineering protein folding degenerated libraries polymerase chain reaction binding assays 6xHis-tag affinity purification