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

Anna V. Hine, Mohammed Ashraf, Laura Frigotto, Matthew E. Smith, Seema Patel, Marcus D. Hughes, Andrew J. Poole, Husam R.M. Hebaishi, Christopher G. Ullman

Research output: Contribution to conferencePaper

Abstract

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering.
Original languageEnglish
PagesP012
Number of pages1
Publication statusPublished - Jul 2013
EventProtein engineering: new approaches and applications - University of Chester, Chester, United Kingdom
Duration: 10 Apr 201312 Apr 2013

Conference

ConferenceProtein engineering: new approaches and applications
CountryUnited Kingdom
CityChester
Period10/04/1312/04/13
OtherA joint Biochemical Society / Protein Society conference. Protein engineering has proved pivotal to our molecular understanding and adaption of proteins by allowing us to interact and manipulate the very coding sequence that underlies folding, structure and function.
Protein engineering is currently undergoing a renaissance with the advent of new technological approaches to address some of major emerging areas in the life sciences, including facilitating the interface with both chemistry and physics. Protein engineering will prove central to areas ranging from synthetic biology to bioprocessing to nanotechnology.
In recent years the traditional idea of simple site directed mutagenesis has been superseded through the developed of new approaches and technologies that have enabled the field of protein engineering to flourish. These new developments that include advanced computational design, development of new and useful biocatalysts, integration of functional biological parts with fabricated devices and construction of next generation biopharmaceuticals. Protein engineering also continues to provide valuable and fundamental understanding of natural protein construction and function that in turn will inevitably improve our ability to generate the next generation of novel proteins.
This meeting will assemble a diverse group of world-leading protein engineers who will highlight the recent advances in our current understanding of how proteins can be tractably manipulated towards an intended function either through rational design or directed evolution.

Fingerprint

Random Allocation
Codon
Mutagenesis
Technology
Amino Acids
Insertional Mutagenesis
Zinc Fingers
Oligonucleotides
Ligation
Molecular Biology
Digestion
Equipment and Supplies
Antibodies
DNA
Enzymes
Proteins

Bibliographical note

Funding: Aston University departmental studentship; Biotechnology and Biological Sciences Research Council [BB/D525756/1]; Aston Research Centre for Healthy Aging (ARCHA) studentship; Technology Strategy Board [720032]

Cite this

Hine, A. V., Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M. D., ... Ullman, C. G. (2013). ProxiMAX randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons. P012. Paper presented at Protein engineering: new approaches and applications, Chester, United Kingdom.
Hine, Anna V. ; Ashraf, Mohammed ; Frigotto, Laura ; Smith, Matthew E. ; Patel, Seema ; Hughes, Marcus D. ; Poole, Andrew J. ; Hebaishi, Husam R.M. ; Ullman, Christopher G. / ProxiMAX randomisation : a new technology for non-degenerate saturation mutagenesis of contiguous codons. Paper presented at Protein engineering: new approaches and applications, Chester, United Kingdom.1 p.
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Hine, AV, Ashraf, M, Frigotto, L, Smith, ME, Patel, S, Hughes, MD, Poole, AJ, Hebaishi, HRM & Ullman, CG 2013, 'ProxiMAX randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons', Paper presented at Protein engineering: new approaches and applications, Chester, United Kingdom, 10/04/13 - 12/04/13 pp. P012.

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

2013. P012 Paper presented at Protein engineering: new approaches and applications, Chester, United Kingdom.

Research output: Contribution to conferencePaper

TY - CONF

T1 - ProxiMAX randomisation

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

AU - Hine, Anna V.

AU - Ashraf, Mohammed

AU - Frigotto, Laura

AU - Smith, Matthew E.

AU - Patel, Seema

AU - Hughes, Marcus D.

AU - Poole, Andrew J.

AU - Hebaishi, Husam R.M.

AU - Ullman, Christopher G.

N1 - Funding: Aston University departmental studentship; Biotechnology and Biological Sciences Research Council [BB/D525756/1]; Aston Research Centre for Healthy Aging (ARCHA) studentship; Technology Strategy Board [720032]

PY - 2013/7

Y1 - 2013/7

N2 - Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering.

AB - Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering.

UR - http://www.biochemistry.org/Portals/0/Conferences/abstracts/SA143/SA143P012.pdf

M3 - Paper

SP - P012

ER -

Hine AV, Ashraf M, Frigotto L, Smith ME, Patel S, Hughes MD et al. ProxiMAX randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons. 2013. Paper presented at Protein engineering: new approaches and applications, Chester, United Kingdom.