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

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

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  • Research Output

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

    Hine, A. V., Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M. D., Poole, A. J., Hebaishi, H. R. M. & Ullman, C. G., Jul 2013, p. P012. 1 p.

    Research output: Contribution to conferencePaper

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

    Hine, A., Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M., Poole, A. J., Hebaishi, H. & Ullman, C. G., Nov 2013.

    Research output: Contribution to conferenceAbstract

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

    Hine, A., Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M., Poole, A. J., Hebaishi, H. & Ullman, C. G., 2013.

    Research output: Contribution to conferencePoster

  • Cite this

    Ashraf, M., Frigotto, L., Smith, M. E., Patel, S., Hughes, M. D., Poole, A. J., Hebaishi, H. R. M., Ullman, C. G., & 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