Journal
BIOCHEMICAL SOCIETY TRANSACTIONS
Volume 41, Issue -, Pages 1189-U250Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BST20130123
Keywords
antibody engineering; gene library; MAX randomization; overlap PCR; protein engineering; saturation mutagenesis
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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]
- Biotechnology and Biological Sciences Research Council [1090346, BB/D525756/1] Funding Source: researchfish
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Back in 2003, we published 'MAX' randomization, 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' randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an a-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' randomization, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomization uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialized chemistry, reagents or 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 predefined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomization is particularly relevant to antibody engineering.
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