High-throughput mutagenesis using a two-fragment PCR approach

Franziska M Heydenreich,Dmitry B Veprintsev,Roger Benoit,Dalibor Milić,Rolf Jaussi,Tamara Miljuš

doi:10.1038/s41598-017-07010-4

Franziska M Heydenreich, Dmitry B Veprintsev + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-07010-4

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Abstract

Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments. We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.

Highlights

Scanning mutagenesis, the replacement of selected single amino acid residues by alanine or any other amino acid[1, 2] is a valuable tool for systematically probing protein functionality
We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of the resulting fragments
The two vector fragments generated in separate PCRs are combined in the simple and efficient Gibson assembly[14], merits of which were extensively discussed by Benoit et al.[15]

Summary

Introduction

The replacement of selected single amino acid residues by alanine or any other amino acid[1, 2] is a valuable tool for systematically probing protein functionality. The rest of the missing V2R mutants (3, 0.8%) were obtained by repeating the PCR with two mutagenic primers in the same reaction mixture (Fig. 1), followed by in vivo recombination in the E. coli Mach[1] strain[22], according to our earlier one-fragment approach[5, 13].

Results

Conclusion