Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering

Abstract

Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. There is thus a need for robust and unbiased molecular biological approaches for the construction of the requisite comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and comprehensive, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple protocol.

Keywords: directed evolution; fluorescence thermostability; iLOV; protein mutagenesis; recombineering.

Figure 1

Plasmid Recombineering (PR) of iLOV generates a specific and comprehensive mutation library. (A) Cartoon of comprehensive PR accomplished using synthetic oligonucleotides tiled across the target gene. (B) Frequency of single amino acid mutations mapped by residue and location in the Round 1 library. Black indicates the WT iLOV residue. White indicates no detected reads. (C) Cartoon of programmable PR targeting a specific sequence space. Sampling of the highest fitness mutants can inform subsequent library design, with recombineering oligos specifically targeting mutations of interest. (D) Mutational distribution of the library after additional rounds of recombineering.

Figure 2

The recombineering mechanism produces moderate bias within the library and can be manipulated by varying the delivered oligonucleotide concentration. (A) Frequency of single amino acid mutations across iLOV in the Round 1 library. (B) Biological replicate of Round 1 library with normalized oligonucleotide concentrations. (C) Observed vs. expected distribution of one, two, or three nucleotide mismatches among single amino acid mutations. The 95% confidence intervals for these measurements are all smaller than +/− 0.0025 (normal approximation to the binomial distribution). (D) Frequency of double mutations in the Round 5 library. White = not detected. Highly represented rows and columns arise from positional bias (Supp. Figure 8). (E) Frequency of double mutations in the Round 5 library as a function of the pairwise distance between double mutations. For each distance, data has been normalized for the number of possible double mutations. Error bars, standard deviation.

Figure 3

A plate-based thermostability screen identifies mutations that improve iLOV fluorescence at elevated temperatures. (A) Cartoon of the thermostability screen assay and subsequent hit validation procedure. (B) Representative fluorescent images of library colonies before and after temperature challenge. (C) Representative fluorescent thermal melt curves of lysate for two thermostable hits. (D) Histogram of T_ms for 93 thermostabilized iLOV variants.

Figure 4

Multiplexing thermostabilizing mutations rapidly identifies doubly improved iLOV variants. (A) 25 thermostabilizing mutations mapped to the iLOV protein sequence. (B) Histogram of initial thermostable hits (Blue) superimposed on hits from the multiplexed library (Cyan). (C) Representative melt curves of two thermostabilized variants compared to iLOV. (D) Crystal structure of iLOV (PDB 4EES) indicating the location of four mutations found in tLOV: R11S, I35V, R90F, G106V.