TITLE

Fitness Loss and Library Size Determination in Saturation Mutagenesis

AUTHOR(S)
Nov, Yuval
PUB. DATE
July 2013
SOURCE
PLoS ONE;Jul2013, Vol. 8 Issue 7, p1
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Saturation mutagenesis is a widely used directed evolution technique, in which a large number of protein variants, each having random amino acids in certain predetermined positions, are screened in order to discover high-fitness variants among them. Several metrics for determining the library size (the number of variants screened) have been suggested in the literature, but none of them incorporates the actual fitness of the variants discovered in the experiment. We present the results of an extensive simulation study, which is based on probabilistic models for protein fitness landscape, and which investigates how the result of a saturation mutagenesis experiment – the fitness of the best variant discovered – varies as a function of the library size. In particular, we study the loss of fitness in the experiment: the difference between the fitness of the best variant discovered, and the fitness of the best variant in variant space. Our results are that the existing criteria for determining the library size are conservative, so smaller libraries are often satisfactory. Reducing the library size can save labor, time, and expenses in the laboratory.
ACCESSION #
89627104

 

Related Articles

  • Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis. Valetti, Francesca; Gilardi, Gianfranco // Biomolecules (2218-273X);Dec2013, Vol. 3 Issue 4, p778 

    Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of...

  • Narrowing Laccase Substrate Specificity Using Active Site Saturation Mutagenesis. Gupta, Nirupama; Farinas, Edgardo T. // Combinatorial Chemistry & High Throughput Screening;Mar2009, Vol. 12 Issue 3, p269 

    The laccase CotA from Bacillus subtilis was converted from a generalist, an enzyme with broad specificity, to a specialist, an enzyme with narrowed specificity. Laccases are members of the multicopper oxidase family and have many applications in biotechnology. To date, it has not been...

  • A novel iterative strategy for protein design. Rossi, Andrea; Maritan, Amos; Micheletti, Cristian // Journal of Chemical Physics;1/22/2000, Vol. 112 Issue 4 

    We propose and discuss a novel strategy for protein design. The method is based on recent theoretical advancements which showed the importance to treat carefully the conformational free energy of designed sequences. In this work we show how computational cost can be kept to a minimum by...

  • Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Ness, Jon E.; Kim, Seran; Gottman, Andrea; Pak, Rob; Krebber, Anke; Borchert, Torben V.; Govindarajan, Sridhar; Mundorff, Emily C.; Minshull, Jeremy // Nature Biotechnology;Dec2002, Vol. 20 Issue 12, p1251 

    We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database...

  • ENGINEERING OF MICROBIAL PROTEASES: IMPROVING STABILITY AND CATALYTIC PERFORMANCES. Mienda, Bashir Sajo; Yahya, Adibah // IIOAB Journal;Nov/Dec2011, Vol. 2 Issue 8, p10 

    Proteolytic enzymes are becoming more ubiquitous as bacteria and fungi. Some proteases from microbial sources are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities in solvents and temperature at industrial scales in order to suit their...

  • Advances In Protein Stability. Arora, Neha; Bhateja, Abhinav; Garg, Tarun; Bilandi, Ajay // Journal of Pharmacy Research;2012, Vol. 5 Issue 4, p2072 

    Chemical modification and protein engineering, by searching for homolog's in hyperthermophiles especially are useful tools for thermo stabilizing proteins, and also for elucidating the mechanism of protein stability that a single or few amino acid replacement(s) in a protein is/are sufficient to...

  • Selection of high-affinity Centyrin FN3 domains from a simple library diversified at a combination of strand and loop positions. Diem, Michael D.; Hyun, Linus; Yi, Fang; Hippensteel, Randi; Kuhar, Elise; Lowenstein, Cassandra; Swift, Edward J.; O'Neil, Karyn T.; Jacobs, Steven A. // PEDS: Protein Engineering, Design & Selection;Oct2014, Vol. 27 Issue 10, p419 

    Alternative scaffold molecules represent a class of proteins important to the study of protein design and mechanisms of protein–protein interactions, as well as for the development of therapeutic proteins. Here, we describe the generation of a library built upon the framework of a...

  • Engineering highly thermostable xylanase variants using an enhanced combinatorial library method. Hokanson, Craig A.; Cappuccilli, Guido; Odineca, Tatjana; Bozic, Marino; Behnke, Craig A.; Mendez, Michael; Coleman, William J.; Crea, Roberto // PEDS: Protein Engineering, Design & Selection;Aug2011, Vol. 24 Issue 8, p597 

    A new directed evolution method was used to enhance the thermostability of the wild-type GH11 xylanase 2 (known as BD-11) from Hypocrea jecorina (Trichoderma reesei). Both Look-Through Mutagenesis (LTMâ„¢), which is a method for rapidly screening selected positions in the protein sequence...

  • An Improved Profile-Level Domain Linker Propensity Index for Protein Domain Boundary Prediction. Yanfeng Zhang; Bin Liu; Qiwen Dong; Victor X. Jin // Protein & Peptide Letters;Jan2011, Vol. 18 Issue 1, p7 

    No abstract available.

Share

Read the Article

Courtesy of THE LIBRARY OF VIRGINIA

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics