ProxiMAX randomisation achieves saturation mutagenesis of contiguous codons without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, it uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents and as such, requires no specialised chemistry, reagents nor equipment. When particular residues are known to affect protein activity/specificity, their combinatorial replacement with all 20 amino acids, or a subset thereof, can provide a rapid route to generating proteins with desirable characteristics. Conventionally, saturation mutagenesis replaced key codons with degenerate ones. Although simple to perform, that procedure resulted in unnecessarily large libraries, termination codons and inherent uneven amino acid representation. ProxiMAX randomisation is an enzyme-based technique that can encode unbiased representation of all or selected amino acids or else can provide required codons in pre-defined ratios. Each saturated position can be defined independently of the others. ProxiMAX randomisation is achieved via saturation cycling: an iterative process comprising blunt end ligation, amplification and digestion with a Type IIS restriction enzyme. We demonstrate both unbiased saturation of a short 6-mer peptide and saturation of a hypervariable region of a scfv antibody fragment, where 11 contiguous codons are saturated with selected codons, in pre-defined ratios. As such, ProxiMAX randomisation is particularly relevant to antibody engineering. The development of ProxiMAX randomisation from concept to reality is described.
|Publication status||Published - 2013|
|Event||Protein and antibody engineering summit - World Trade Center, Boston, United States|
Duration: 29 Apr 2013 → 3 May 2013
|Conference||Protein and antibody engineering summit|
|Abbreviated title||PEGS 2014|
|Period||29/04/13 → 3/05/13|
Hine, A., Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M., Poole, A. J., Hebaishi, H., & Ullman, C. G. (2013). ProxiMAX Randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons. Poster session presented at Protein and antibody engineering summit, Boston, United States.