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uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

View ORCID ProfileMason J. Appel, View ORCID ProfileScott A. Longwell, Maurizio Morri, Norma Neff, View ORCID ProfileDaniel Herschlag, View ORCID ProfilePolly M. Fordyce
doi: https://doi.org/10.1101/2021.08.04.455146
Mason J. Appel
1Department of Biochemistry, Stanford University, Stanford, CA 94305
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Scott A. Longwell
2Department of Bioengineering, Stanford University, Stanford, CA 94305
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Maurizio Morri
3Chan Zuckerberg Biohub, San Francisco, CA 94110
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Norma Neff
3Chan Zuckerberg Biohub, San Francisco, CA 94110
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Daniel Herschlag
1Department of Biochemistry, Stanford University, Stanford, CA 94305
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  • For correspondence: herschla@stanford.edu pfordyce@stanford.edu
Polly M. Fordyce
2Department of Bioengineering, Stanford University, Stanford, CA 94305
3Chan Zuckerberg Biohub, San Francisco, CA 94110
4Department of Genetics, Stanford University, Stanford, CA 94305
5ChEM-H Institute, Stanford University, Stanford, CA 94305
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  • For correspondence: herschla@stanford.edu pfordyce@stanford.edu
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ABSTRACT

New high-throughput biochemistry techniques complement selection-based approaches and provide quantitative kinetic and thermodynamic data for thousands of protein variants in parallel. With these advances, library generation rather than data collection has become rate limiting. Unlike pooled selection approaches, high-throughput biochemistry requires mutant libraries in which individual sequences are rationally designed, efficiently recovered, sequence-validated, and separated from one another, but current strategies are unable to produce these libraries at the needed scale and specificity at reasonable cost. Here, we present a scalable, rapid, and inexpensive approach for creating User-designed Physically Isolated Clonal–Mutant (uPIC–M) libraries that utilizes recent advances in oligo synthesis, high-throughput sample preparation, and next-generation sequencing. To demonstrate uPIC–M, we created a scanning mutant library of SpAP, a 541 amino acid alkaline phosphatase, and recovered 94% of desired mutants in a single iteration. uPIC–M uses commonly available equipment and freely downloadable custom software and can produce a 5000 mutant library at 1/3 the cost and 1/5 the time of traditional techniques.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://osf.io/k3rjy/

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted August 04, 2021.
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uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry
Mason J. Appel, Scott A. Longwell, Maurizio Morri, Norma Neff, Daniel Herschlag, Polly M. Fordyce
bioRxiv 2021.08.04.455146; doi: https://doi.org/10.1101/2021.08.04.455146
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uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry
Mason J. Appel, Scott A. Longwell, Maurizio Morri, Norma Neff, Daniel Herschlag, Polly M. Fordyce
bioRxiv 2021.08.04.455146; doi: https://doi.org/10.1101/2021.08.04.455146

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