ProxiMAX Randomisation

a new technology for non-degenerate saturation mutagenesis of contiguous codons

Anna Hine, M. Ashraf, Laura Frigotto, Matthew E Smith, Seema Patel, Marcus Hughes, Andrew James Poole, Husam Hebaishi, Christopher G. Ullman

Research output: Contribution to conferencePoster

Abstract

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.
Original languageEnglish
Publication statusPublished - 2013
EventProtein and antibody engineering summit - World Trade Center, Boston, United States
Duration: 29 Apr 20133 May 2013

Conference

ConferenceProtein and antibody engineering summit
Abbreviated titlePEGS 2014
CountryUnited States
CityBoston
Period29/04/133/05/13

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Random Allocation
Codon
Mutagenesis
Technology
Amino Acids
Single-Chain Antibodies
Immunoglobulin Fragments
Terminator Codon
Enzymes
Oligonucleotides
Libraries
Ligation
Molecular Biology
Digestion
Proteins
Equipment and Supplies
Peptides
Antibodies

Cite this

Hine, A., Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M., ... 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.
Hine, Anna ; Ashraf, M. ; Frigotto, Laura ; Smith, Matthew E ; Patel, Seema ; Hughes, Marcus ; Poole, Andrew James ; Hebaishi, Husam ; Ullman, Christopher G. / 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.
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abstract = "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.",
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Hine, A, Ashraf, M, Frigotto, L, Smith, ME, Patel, S, Hughes, M, Poole, AJ, Hebaishi, H & Ullman, CG 2013, 'ProxiMAX Randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons' Protein and antibody engineering summit, Boston, United States, 29/04/13 - 3/05/13, .

ProxiMAX Randomisation : a new technology for non-degenerate saturation mutagenesis of contiguous codons. / Hine, Anna; Ashraf, M.; Frigotto, Laura; Smith, Matthew E; Patel, Seema; Hughes, Marcus; Poole, Andrew James; Hebaishi, Husam; Ullman, Christopher G.

2013. Poster session presented at Protein and antibody engineering summit, Boston, United States.

Research output: Contribution to conferencePoster

TY - CONF

T1 - ProxiMAX Randomisation

T2 - a new technology for non-degenerate saturation mutagenesis of contiguous codons

AU - Hine, Anna

AU - Ashraf, M.

AU - Frigotto, Laura

AU - Smith, Matthew E

AU - Patel, Seema

AU - Hughes, Marcus

AU - Poole, Andrew James

AU - Hebaishi, Husam

AU - Ullman, Christopher G.

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

M3 - Poster

ER -

Hine A, Ashraf M, Frigotto L, Smith ME, Patel S, Hughes M et al. ProxiMAX Randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons. 2013. Poster session presented at Protein and antibody engineering summit, Boston, United States.