Protein Engineering Protocols - Mycobacteriology research center

Synthesis of Libraries and Screening With the DHFR PCA 261In a protein-engineering project in which the model system under study isvery well-characterized, only controls 1, 2, 4, and 6 are essential for establishingthe specificity and stringency of the assay. In the case of the RBD–ras interaction,a comprehensive mutagenesis study and its effect on the K dfor bindingof the RBD had already been published (46). These data permitted us to engineerseveral mutants that reduce the K dfor association of RBD–ras more thanthree orders of magnitude (see, e.g., Fig. 3). These mutants and others weretested in the DHFR PCA, allowing us to establish that the assay is able to detectbinding for the RBD to ras for mutants with a K don the order of 1 µM. In addition,published mutants that destabilize the protein fold, such as core hydrophobicresidues (valine, leucine, or isoleucine) side-chain truncation to alanine,could be used as a stringency test.3.2. Library Synthesis1. To have a nonbiased library, we first generated a template in which the region to bevaried was deleted and replaced by a stop codon, inserting also a frame shift and aunique restriction site allowing for its unequivocal identification (see Note 1).2. To generate each library, we synthesized two PCR products that partially overlap(typically a 18–20 basepair [bp] hybridization region). For example, for PCR 1,we used one primer hybridizing in the promoter region of our vector (120 bpupstream of the start codon; see Fig. 4) and one primer hybridizing in the regionimmediately 5′ of the section targeted for degeneracy. For PCR 2, we used onetwo-arms oligonucleotide and a primer that hybridizes to the F [1,2] (120 bpdownstream of the 3′-end of the open-reading frame; see Fig. 4). Typically, thePCR program was set as following: 1 min hot start at 94°C, 25 cycles of 20 s at94°C, 30 s at 52°C, and 30 s at 72°C (see Note 2). Finally, the reactions were runfor 10 min at 72°C to ensure completion of the elongation.3. The PCR products are analyzed on an agarose gel. If the desired product is obtained,the remainder of the PCR product is loaded on a gel. We have advantageously usedGelstar and the Dark Reader (see Note 3) to visualize PCR products on agarosegels. It permits observation of bands under blue light (400–500 nm), wavelengthsthat do not damage DNA, in contrast to ultraviolet light (this allows one to cut thebands out and to proceed easily, in parallel, to the generation of several libraries).4. Next, the bands are gel purified with Qiaex II (see Note 4).5. Approximately 300 ng of the PCR product from PCR 1 and 2 are combined (seeFig. 4 and Note 5) with 0.2 µM of the terminal primers (hybridizing in the promoterand in F [1,2]) that anneal in regions 5′ and 3′, respectively, to the product ofPCR 1 and 2. The PCR 3 program is the following: 1 min hot-start at 94°C, 10cycles of 20 s at 94°C, 30 s at 52°C, and 30 s at 72°C. Finally, 10 min at 72°C toensure completion of the elongation (see Note 6).6. The entry vector pQE-32 ∆ F [1,2] (see Note 7) and the resultant PCR productsare digested with the appropriate restriction enzyme (SphI and XhoI, in this case).7. Bands are purified according to Subheading 3.2., step 4.