Saturation mutagenesis and precision libraries: the numbers game

Anna V. Hine, Anupama Chembath, Marta Ferreira Amaral, Ben Wagstaffe, Yvonne Stark, Andreas Plückthun

Research output: Contribution to conferenceAbstract

Abstract

Saturation mutagenesis is a vital tool in the protein engineer’s arsenal, but involves astronomical numbers of genes when targeting multiple codons. Some argue that an iterative codon-by-codon approach is the answer, whilst others are convinced that owing to context-dependency, iteration will exclude fundamental best “hits”. Meanwhile, some advocate creating vast “sampling” libraries while others champion full representation. Whatever the stance, the numbers game in saturation mutagenesis plays a vital role in the eventual outcome of a protein engineering project.
Embracing minimalist, non-degenerate saturation involves extra work up-front, but minimizes the numbers effect during the most intensive part of the whole process – namely screening the resulting libraries. Appropriate non-degenerate saturation can eliminate undesirable residues prone to oxidation or helix disruption or can reduce the frequency of undesirable dipeptide occurrences, while much smaller ligand-specific libraries can be created exclusively. Different applications such as antibody loops or active sites of enzymes require different synthetic protocols to generate non-degenerate saturation libraries. This presentation will examine the numbers, the various platform technologies created in our lab over the last decade, whether proprietary or open access, consecutive or disperse codons, manual or automated synthesis and the quality of resulting hits that require no further affinity maturation.
Original languageEnglish
Publication statusPublished - 2019
EventProtein engineering II: from new molecules to new processes - York, United Kingdom
Duration: 15 Jul 201917 Jul 2019
https://www.eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID=17614&ef_sel_menu=219&eventID=42

Conference

ConferenceProtein engineering II: from new molecules to new processes
CountryUnited Kingdom
Period15/07/1917/07/19
Internet address

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engineering

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Hine, A. V., Chembath, A., Ferreira Amaral, M., Wagstaffe, B., Stark, Y., & Plückthun, A. (2019). Saturation mutagenesis and precision libraries: the numbers game. Abstract from Protein engineering II: from new molecules to new processes, United Kingdom.
Hine, Anna V. ; Chembath, Anupama ; Ferreira Amaral, Marta ; Wagstaffe, Ben ; Stark, Yvonne ; Plückthun, Andreas . / Saturation mutagenesis and precision libraries: the numbers game. Abstract from Protein engineering II: from new molecules to new processes, United Kingdom.
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title = "Saturation mutagenesis and precision libraries: the numbers game",
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note = "Protein engineering II: from new molecules to new processes ; Conference date: 15-07-2019 Through 17-07-2019",
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Hine, AV, Chembath, A, Ferreira Amaral, M, Wagstaffe, B, Stark, Y & Plückthun, A 2019, 'Saturation mutagenesis and precision libraries: the numbers game' Protein engineering II: from new molecules to new processes, United Kingdom, 15/07/19 - 17/07/19, .

Saturation mutagenesis and precision libraries: the numbers game. / Hine, Anna V.; Chembath, Anupama; Ferreira Amaral, Marta; Wagstaffe, Ben; Stark, Yvonne ; Plückthun, Andreas .

2019. Abstract from Protein engineering II: from new molecules to new processes, United Kingdom.

Research output: Contribution to conferenceAbstract

TY - CONF

T1 - Saturation mutagenesis and precision libraries: the numbers game

AU - Hine, Anna V.

AU - Chembath, Anupama

AU - Ferreira Amaral, Marta

AU - Wagstaffe, Ben

AU - Stark, Yvonne

AU - Plückthun, Andreas

PY - 2019

Y1 - 2019

N2 - Saturation mutagenesis is a vital tool in the protein engineer’s arsenal, but involves astronomical numbers of genes when targeting multiple codons. Some argue that an iterative codon-by-codon approach is the answer, whilst others are convinced that owing to context-dependency, iteration will exclude fundamental best “hits”. Meanwhile, some advocate creating vast “sampling” libraries while others champion full representation. Whatever the stance, the numbers game in saturation mutagenesis plays a vital role in the eventual outcome of a protein engineering project.Embracing minimalist, non-degenerate saturation involves extra work up-front, but minimizes the numbers effect during the most intensive part of the whole process – namely screening the resulting libraries. Appropriate non-degenerate saturation can eliminate undesirable residues prone to oxidation or helix disruption or can reduce the frequency of undesirable dipeptide occurrences, while much smaller ligand-specific libraries can be created exclusively. Different applications such as antibody loops or active sites of enzymes require different synthetic protocols to generate non-degenerate saturation libraries. This presentation will examine the numbers, the various platform technologies created in our lab over the last decade, whether proprietary or open access, consecutive or disperse codons, manual or automated synthesis and the quality of resulting hits that require no further affinity maturation.

AB - Saturation mutagenesis is a vital tool in the protein engineer’s arsenal, but involves astronomical numbers of genes when targeting multiple codons. Some argue that an iterative codon-by-codon approach is the answer, whilst others are convinced that owing to context-dependency, iteration will exclude fundamental best “hits”. Meanwhile, some advocate creating vast “sampling” libraries while others champion full representation. Whatever the stance, the numbers game in saturation mutagenesis plays a vital role in the eventual outcome of a protein engineering project.Embracing minimalist, non-degenerate saturation involves extra work up-front, but minimizes the numbers effect during the most intensive part of the whole process – namely screening the resulting libraries. Appropriate non-degenerate saturation can eliminate undesirable residues prone to oxidation or helix disruption or can reduce the frequency of undesirable dipeptide occurrences, while much smaller ligand-specific libraries can be created exclusively. Different applications such as antibody loops or active sites of enzymes require different synthetic protocols to generate non-degenerate saturation libraries. This presentation will examine the numbers, the various platform technologies created in our lab over the last decade, whether proprietary or open access, consecutive or disperse codons, manual or automated synthesis and the quality of resulting hits that require no further affinity maturation.

M3 - Abstract

ER -

Hine AV, Chembath A, Ferreira Amaral M, Wagstaffe B, Stark Y, Plückthun A. Saturation mutagenesis and precision libraries: the numbers game. 2019. Abstract from Protein engineering II: from new molecules to new processes, United Kingdom.