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Journal of Biomolecular Screening
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Article

Simulation Modeling of Pooling for Combinatorial Protein Engineering

Karen M. Polizzi1, Cody U. Spencer2, Anshul Dubey1, Ichiro Matsumura3, Jay H. Lee1, Matthew J. Realff1, Andreas S. Bommarius1*

1 School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA.
2 College of Computing, Georgia Institute of Technology, Atlanta, GA.
3 Department of Biochemistry, School of Medicine, Emory University, Atlanta, GA.

* To whom correspondence should be addressed. E-mail: andreas.bommarius{at}chbe.gatech.edu.


  Abstract

Pooling in directed-evolution experiments will greatly increase the throughput of screening systems, but important parameters such as the number of good mutants created and the activity level increase of the good mutants will depend highly on the protein being engineered. The authors developed and validated a Monte Carlo simulation model of pooling that allows the testing of various scenarios in silico before starting experimentation. Using a simplified test system of 2 enzymes, {beta}galactosidase (supermutant, or greatly improved enzyme) and {beta}-glucuronidase (dud, or enzyme with ancestral level of activity), the model accurately predicted the number of supermutants detected in experiments within a factor of 2. Additional simulations using more complex activity distributions show the versatility of the model. Pooling is most suited to cases such as the directed evolution of new function in a protein, where the background level of activity is minimized, making it easier to detect small increases in activity level. Pooling is most successful when a sensitive assay is employed. Using the model will increase the throughput of screening procedures for directed-evolution experiments and thus lead to speedier engineering of proteins.

Key Words: directed evolution, high-throughput screening, Monte Carlo simulation, protein engineering

First published on October 18, 2005, doi:10.1177/1087057105280134

Journal of Biomolecular Screening 2005;10:856.

A more recent version of this article appeared on December 1, 2005

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