Protein Engineering Protocols - Mycobacteriology research center

252 Campbell-Valois and Michnickfolds rapidly to the correct structure and is sufficiently stable, it should interactwith its natural binding partner, the small GTPase, ras. Fusing the RBDlibrary to one complementary fragment of DHFR and ras to the other, and thenco-expressing these in E. coli, grown under selective pressure as described earlierin this section, it can be reasoned that fast-folding and stable members of theRBD library will interact with ras and allow for the reconstitution of DHFRactivity and the rescue of cell growth. We chose an efficient and realistic way todesign libraries that allowed for exploring the maximum sequence diversity in ameaningful way, while creating libraries of reasonable size. Based on the questionswe chose to address in these studies, a meaningful way to explore sequencespace is to generate libraries in which only a small stretch of contiguous residuesare varied at a time (10). Examination of the RBD structure allows one to dissectit into 13 regions corresponding to individual β-turns or loops, β-strands, andone α-helix (two libraries were generated for this region corresponding to aminoand carboxyl termini of the helix), ranging in length from four to eight aminoacids (41,42). On this basis, we created 13 degenerated libraries, in which eachwild-type codon is replaced by a NNK codon (where N is any nucleotide, andK is G or T) that allows the insertion of the 20 amino acids at each varied positionin the sequence. These libraries were screened for binding to ras by theDHFR PCA in E. coli. In addition to the screening being performed entirely invivo, a key advantage of this approach is that expressed RBD library membersthat interact well with ras can be purified for physical analysis without having toswitch to another expression system.Herein, we present the protocols and proposed trouble-shooting strategies,based on the technical challenges that we have encountered in the design andsynthesis of the degenerated libraries and in their screening with the DHFRPCA. Hopefully, these protocols are general enough to be useful not only tothose interested in folding, but more generally to problems requiring the optimizationof protein–protein interactions.2. Materials2.1. Library Synthesis1. Oligonucleotide primers (IDT). The primers with positions at which multiplebases are allowed are hand mixed to assure that the desired ratio of each base isrespected. These are sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) purified.2. Taq polymerase (Fermentas).3. Agarose gel: agarose (Bioshop) Dark Reader (Clare Chemical Research) andGelstar (Biowittaker Molecular Applications).4. Gel purification, QIAEX II or, preferably, QIAquick gel extraction kit(Qiagen).