Chapter title |
Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments
|
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Chapter number | 15 |
Book title |
Enzyme Engineering
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Published in |
Methods in molecular biology, January 2013
|
DOI | 10.1007/978-1-62703-293-3_15 |
Pubmed ID | |
Book ISBNs |
978-1-62703-292-6, 978-1-62703-293-3
|
Authors |
Sandeep Chakraborty, Renu Minda, Lipika Salaye, Abhaya M. Dandekar, Swapan K. Bhattacharjee, Basuthkar J. Rao, Chakraborty, Sandeep, Minda, Renu, Salaye, Lipika, Dandekar, Abhaya M., Bhattacharjee, Swapan K., Rao, Basuthkar J. |
Abstract |
Error-prone PCR, DNA shuffling, and saturation mutagenesis are techniques used by protein engineers to mimic the natural "evolutionary walk" that conjures new enzymes. Rational design is often critical in efforts to accelerate this "random walk" into a "resolute sprint." Previous work by our group established a computational method for detecting active sites (CLASP) based on spatial and electrostatic properties of catalytic residues, and a method to quantify promiscuous activities in a wide range of proteins (PROMISE). Here, we describe a rational design flow (DECAAF) based on the PROMISE methodology to choose a protein which, when subjected to minimal mutations, is most likely to mirror the scaffold of a desired enzymatic function. Modeling the diversity in catalytic sites and providing precise user control to guide the search is a key goal of our implementation. The flow details have been worked out in a real-life example to select a plant protein to substitute for human neutrophil elastase in a chimeric antimicrobial enzyme designed to bolster the innate immune defense system in plants. |
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