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.
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.
Campbell-Valois
François-Xavier
FX
Département de Biochimie, Université de Montréal, Québec, Canada.
Michnick
Stephen W
SW
D002846:Chromatography, Affinity; D015723:Gene Library; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry*
D009942:Organometallic Compounds; D019151:Peptide Library; C088321:nickel nitrilotriacetic acid; D013762:Tetrahydrofolate Dehydrogenase; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D009571:Nitrilotriacetic Acid