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
SN - 0300-5127
VL - 41
SP - 1189
EP - 1194
JO - Biochemical Society Transactions
JF - Biochemical Society Transactions
IS - 5
M1 - BST2013/0123
ER -