Protein Engineering Protocols - Mycobacteriology research center

Synthesis of Libraries and Screening With the DHFR PCA 251(refs. 31 and 32; reviewed in refs. 33 and 34). The PCA strategy relies on theassociation and folding of a reporter protein or enzyme from fragments, drivenby the interaction of two proteins to which the fragments are fused. The reconstitutionof the reporter protein fold and, thus, detectable catalytic activity,depends on the interaction of the fused proteins. In particular, a simple survivalselectionassay has been developed for screening libraries in Escherichia coli,based on the murine dihydrofolate reductase (mDHFR) as the reporter PCA (32).In E. coli, as in all prokaryotes and eukaryotes, the DHFR product, tetrahydrofolate,is necessary for the synthesis of thymine, glycine, serine, and adenine,whereas, in prokaryotes, it is also required for synthesis of pantothenate. DHFRactivity is, thus, absolutely required for cell growth and division in the absenceof a source of DHFR end products. E. coli can be made dependant on expressionof recombinant mDHFR by treatment of the cells with trimethoprim, a folateanalog that is 12,000 times more potent an inhibitor of E. coli than mammalianDHFRs (35). Thus, the principle of the mDHFR PCA is that two proteins fused tocomplementary fragments of mDHFR must be coexpressed and interact togetherin E. coli grown in minimal (M9) medium supplemented with trimethoprim forcells to grow and divide (31). In a first demonstration of a library-against-libraryscreen, the DHFR PCA was used to identify optimally heterodimerizing pairs ofleucine zipper-forming sequences from individual libraries containing 6 × 10 10possible combinations of sequences. Competition experiments and “library shuffling”strategies were devised to improve library screening coverage, to furtheroptimize dimerizing pairs, and, finally, to identify a “winner pair” (32,36). Morerecently, DHFR PCA was adapted for screening and selection of single-chainantibodies in vivo (37). The all-in-one genetic screening approach of the DHFRPCA strategy is the key feature allowing for simple performance of libraryagainst-libraryscreening, because selective pressure is applied concomitantlyon both library populations during several cycles, without the tedious alternationbetween discrete expansion and screening steps associated with phagedisplay. Thus, PCA truly allows for the study of sequences covariation ofoligomeric partners.The results obtained with leucine zippers convinced us that the assay could beuseful for tackling problems that are more challenging. In the zipper studies,only a handful of key amino acid positions were varied, and only between twoand four amino acid substitutions were allowed. Based on previous theoreticalwork (38), we have attempted to rigorously and exhaustively determine thesequence determinants for folding of the ras-binding domain (RBD) of the serine/threonineprotein kinase, raf (39). The premise of our approach is similar toa previously published strategy aimed at identifying sequences that support rapidfolding and stability of proteins selected by phage display (40). The principle, asapplied to the raf RBD, is as follows: if the sequence of a given RBD variant