Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Abstract

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change

“…For PEGylation experiments, the pTK-SM N100-GFP-V5 (WT) (1) was used to generate a cysteine-free mutant, SM N100 C31S,C46S (C-null) and C46S or C31S mutants (designated 31C and 46C, respectively). The C-null plasmid was used to generate single cysteine mutants with either T3C, T9C, T11C, G18C, S37C, V41C, S43C, S59C, G61C, L65C, S67C, S71C, S83C, or S87C mutations using megaprimer site-directed mutagenesis (11). The pCMV-Insig6xmyc (Insig-myc) (12) plasmid used for differential solubilization assays was a generous gift from Drs Michael S. Brown and Joseph L. Goldstein (University of Texas Southwestern, Dallas, TX).…”

A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis

“…We used megaprimer sitedirected mutagenesis (41) to mutate pTK-SM N100-GFP-V5 and pTK-SM-V5 to generate 49 alanine mutants as indicated in the figures. The pTK-SM N49-GFP-V5 construct was generated previously (8).…”

Directed Evolution of an Enantioselective Enoate‐Reductase: Testing the Utility of Iterative Saturation Mutagenesis

“…In the present study we applied a "short-cut" quality control as reported earlier. [28] The quality was checked by performing sequence analyses of pooled plasmids for each library prior to transformation into the expression strain (Experimental Section). The results of such an analysis are shown in Table 1.…”