The primer used for megaprimer generation can also encode targeted mutations (data not shown)

The primer used for megaprimer generation can also encode targeted mutations (data not shown). MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared to current methods. Keywords:directed evolution, protein engineering, error prone PCR, kunkel mutagenesis, megaprimers == Introduction == Directed evolution, the generation of a library of mutations followed by function-based selection, is a standard tool for many protein engineering, synthetic biology and metabolic engineering applications (1,2). This method has been applied to a variety of problems, including epitope mapping of protein-protein interactions (35), generation of novel protein function (6,7), optimization of metabolic pathways through promoter or transcription factor engineering (8,9) and enzyme engineering, such as modulation of substrate use (10) or generation of novel catalytic activity (11). Approaches to generate libraries of variants include targeted and random mutagenesis in addition to recombination-based methods, such as DNA shuffling (12) and non-templated recombination (13), while still other methods use a combination of these Obatoclax mesylate (GX15-070) techniques (14). Of these, random mutagenesis is a popular and effective tool when combined with a robust function-based screen (15,16) Obatoclax mesylate (GX15-070) and can be used to adjust a variety of protein biochemical or biophysical features, such as antibody affinity for ligands (17). The goal Obatoclax mesylate (GX15-070) of a directed evolution experiment is to identify clones with enhanced function; the probability of success is enhanced by generation of large libraries encoding few wild-type variants. Generation of randomly mutated libraries inE. colitypically proceeds with four steps: (i) amplification of the region of interest under error-prone conditions, (ii) restriction enzyme digestion, (iii) ligation Obatoclax mesylate (GX15-070) into a similarly digested plasmid and (iv) transformation into competent cells. Methods to improve error-prone amplification have been extensively studied (18). For example, PCR conditions can be optimized for library quality, but this has little impact on the final library size. Similarly, as long as highly competentE. coliare used for transformation, this step does not typically limit library size (19). Sequential enzymatic digestion and ligation steps can limit the size of the final library (due to Mouse monoclonal to ER inefficient digestion or elimination/introduction of restriction sites during error-prone PCR (20)), often require optimization of insert:template ratios during ligation (21), and can be both time- and resource-intensive. Techniques designed to avoid this pitfall often require Obatoclax mesylate (GX15-070) altered reaction conditions, specialized primer design or additional time consuming sub-cloning steps (22,23). As a result, the cloning steps present the major bottleneck to library generation. Alternatives to enzymatic digestion and ligation typically employ PCR orin vivomutagenesis (24). Direct DNA manipulation can be avoided byin vivotechniques, such as somatic hypermutation in B cells (25) and plasmid amplification inE. colistrains lacking recombination/repair enzymes (e.g., XL1-Red, Stratagene). However, the use of XL1-Red suffers from a very low mutation rate, requiring propagation of plasmids for up to 100 generations to achieve sufficient error rates, and does not restrict mutations to a region of interest, but introduces them throughout the plasmid and bacterial genome (23). QuikChange (Stratagene) is a widely used method for site-directed mutagenesis and has been expanded for targeted mutagenesis libraries, including those using megaprimers (20,21,2628). Here, primers encoding the desired mutation(s) are annealed to methylated template plasmid. A linear amplification step extends the primer to generate a replica of the entire plasmid. The wild-type template is digested with the methyl-DNA specific enzymeDpnI and,.