A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons

Abstract

Random mutagenesis followed by selection or screening is a commonly used strategy to improve protein function. Despite many available methods for random mutagenesis, nearly all generate mutations at the nucleotide level. An ideal mutagenesis method would allow for the generation of 'codon mutations' to change protein sequence with defined or mixed amino acids of choice. Herein we report a method that allows for mutations of one, two or three consecutive codons. Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences. As a demonstration of the method, we performed multi-codon scanning on the gene encoding superfolder GFP (sfGFP). Characterization of 50 randomly chosen clones from each library showed that more than 40% of the mutants in these three libraries contained seamless, in-frame mutations with low site preference. By screening only 500 colonies from each library, we successfully identified several spectra-shift mutations, including a S205D variant that was found to bear a single excitation peak in the UV region.

PFunkel: Efficient, Expansive, User-Defined Mutagenesis

“…Error-prone PCR suffers from mutational bias, the inability to define the mutational composition, and the inability to effectively cause most amino acid substitutions, which require two or three mutations in a single codon. Methods that rely on random DNA cleavage reagents or transposons for mutating short sequences of DNA suffer from complex procedures and the inability to target the mutations [6]–[8].…”

To get what we aim for – progress in diversity generation methods

“…SeSaM-methods MCST [57] dITP-PCR [62] POE-PCR [63] MegAnneal [60] TRINS [56] epRCA [58] SeSaM-Tv-II [59] SeSaM-III [61] Chemical extraction steps in which significant amounts of DNA are lost [33,35] and/or the need for chemo-or enzymatic DNA modifications, such as phosphorylation [31,35] and biotinylation [34]. Broadly employed focused mutagenesis methods (e.g.…”

“…Random mutagenesis methods have been grouped into three mutagenesis categories: (a) PCR-based; (b) chemical mutagens; and (c) whole cell methods [1,18]. Progress in the last 3 years is summarized by covering multicodon scanning mutagenesis (MCST), 2′-deoxyinosine 5′-triphosphate (dIT)P-PCR, prolonged overlap extension-PCR (POE-PCR), MegAnneal, tandem repeat insertion (TRINS), error-prone multiply-primed rolling circle amplification (epRCA), sequence saturation mutagenesis (SeSaM)-Tv-II and SeSaM-III [56][57][58][59][60][61][62][63] (Fig. 1).…”

“…Progress in the diversity of mutant libraries has been achieved for the MCST [57], MegAnneal [60] and SeSaM [59,61,67] methods by increasing the number of transversions and subsequent mutations. SeSaM outperforms epPCR and transposon-based methods in terms of diversity and at the expense of simplicity, as well as time requirements (Megawhop < epPCR < transposon < SeSaM) for generating a random mutant library.…”

Rational Protein Sequence Diversification by Multi-Codon Scanning Mutagenesis

“…While this is quite easy to do in the lab using methods such as error-prone PCR(1) and/or DNA shuffling(2), such changes are limited by the redundancy of the genetic code. Indeed many nucleotide changes do not result in protein changes and simply dilute out functional diversity.…”