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

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

Research output: Contribution to journalArticle

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
Article numberBST2013/0123
Pages (from-to)1189-1194
Number of pages6
JournalBiochemical Society Transactions
Volume41
Issue number5
Early online date23 Sep 2013
DOIs
Publication statusPublished - Oct 2013

Fingerprint

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

Bibliographical note

© 2013 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

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].

Supplementary online data.

Keywords

  • antibody engineering
  • gene library
  • MAX randomization
  • overlap PCR
  • protein engineering
  • saturation mutagenesis

Cite this

Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M. D., Poole, A. J., ... Hine, A. V. (2013). ProxiMAX randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons. Biochemical Society Transactions, 41(5), 1189-1194. [BST2013/0123]. https://doi.org/10.1042/BST20130123
Ashraf, Mohammed ; Frigotto, Laura ; Smith, Matthew E. ; Patel, Seema ; Hughes, Marcus D. ; Poole, Andrew J. ; Hebaishi, Husam R.M. ; Ullman, Christopher G. ; Hine, Anna V. / ProxiMAX randomisation : a new technology for non-degenerate saturation mutagenesis of contiguous codons. In: Biochemical Society Transactions. 2013 ; Vol. 41, No. 5. pp. 1189-1194.
@article{db62adc0a04849d2b8b8a1e59f24a72c,
title = "ProxiMAX randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons",
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.",
keywords = "antibody engineering, gene library, MAX randomization, overlap PCR, protein engineering, saturation mutagenesis",
author = "Mohammed Ashraf and Laura Frigotto and Smith, {Matthew E.} and Seema Patel and Hughes, {Marcus D.} and Poole, {Andrew J.} and Hebaishi, {Husam R.M.} and Ullman, {Christopher G.} and Hine, {Anna V.}",
note = "{\circledC} 2013 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. 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]. Supplementary online data.",
year = "2013",
month = "10",
doi = "10.1042/BST20130123",
language = "English",
volume = "41",
pages = "1189--1194",
journal = "Biochemical Society Transactions",
issn = "0300-5127",
publisher = "Portland Press Ltd.",
number = "5",

}

Ashraf, M, Frigotto, L, Smith, ME, Patel, S, Hughes, MD, Poole, AJ, Hebaishi, HRM, Ullman, CG & Hine, AV 2013, 'ProxiMAX randomisation: a new technology for non-degenerate saturation mutagenesis of contiguous codons', Biochemical Society Transactions, vol. 41, no. 5, BST2013/0123, pp. 1189-1194. https://doi.org/10.1042/BST20130123

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

In: Biochemical Society Transactions, Vol. 41, No. 5, BST2013/0123, 10.2013, p. 1189-1194.

Research output: Contribution to journalArticle

TY - JOUR

T1 - ProxiMAX randomisation

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

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.

AU - Hine, Anna V.

N1 - © 2013 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. 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]. Supplementary online data.

PY - 2013/10

Y1 - 2013/10

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.

KW - antibody engineering

KW - gene library

KW - MAX randomization

KW - overlap PCR

KW - protein engineering

KW - saturation mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=84884666223&partnerID=8YFLogxK

U2 - 10.1042/BST20130123

DO - 10.1042/BST20130123

M3 - Article

VL - 41

SP - 1189

EP - 1194

JO - Biochemical Society Transactions

JF - Biochemical Society Transactions

SN - 0300-5127

IS - 5

M1 - BST2013/0123

ER -