Abstract
SCHEMA is a method for designing libraries of novel proteins by recombination of homologous sequences. The goal is to maximize the number of folded proteins while simultaneously generating significant sequence diversity. Here, we use the RASPP algorithm to identify optimal SCHEMA designs for shuffling contiguous elements of sequence. To exemplify the method, SCHEMA is used to recombine five fungal cellobiohydrolases (CBH1s) to produce a library of more than 390,000 novel CBH1 sequences.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Voigt CA, Martinez C, Wang Z-G, Mayo SL, Arnold FH (2002) Protein building blocks preserved by recombination. Nat Struct Biol 9:553–558
Meyer M, Hochrein L, Arnold FH (2006) Structure-guided SCHEMA recombination of distantly related β-lactamases. Protein Eng Des Sel 19:563–570
Endelman J, Silberg J, Wang Z, Arnold FH (2004) Site-directed protein recombination as a shortest-path problem. Protein Eng Des Sel 17:589–594
Romero P, Stone E, Lamb C, Chantranupong L, Krause A, Miklos A, Hughes R, Fechtel B, Ellington AD, Arnold FH, Georgiou G (2012) SCHEMA-designed variants of human Arginase I and II reveal sequence elements important to stability and catalysis. ACS Synth Biol 1:221–228
Li Y, Drummond DA, Sawayama AM, Snow CD, Bloom JD, Arnold FH (2007) A diverse family of thermostable cytochrome P450s created by recombination of stabilizing fragments. Nat Biotechnol 25:1051–1056
Heinzelman P, Komor R, Kanaan A, Romero PA, Yu X, Mohler S, Snow C, Arnold FH (2010) Efficient screening of fungal cellobiohydrolase class I enzymes for thermostabilizing sequence blocks by SCHEMA structure-guided recombination. Protein Eng Des Sel 23:871–880
Otey CR, Landwehr M, Endelman JB, Hiraga K, Bloom JD, Arnold FH (2006) Structure-guided recombination creates an artificial family of cytochromes P450. PLoS Biol 4:e112
Heinzelman P, Romero PA, Arnold FH (2013) Efficient sampling of SCHEMA chimera families for identification of useful sequence elements. In: Keasling A (ed) Methods in enzymology: methods in protein design. Elsevier Ltd, Oxford, UK
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948
Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen M-Y, Pieper U, Sali A (2007) Comparative protein structure modeling using MODELLER. Curr Protoc Protein Sci 50:2.9.1–2.9.31
Hiraga K, Arnold FH (2003) General method for sequence-independent site-directed chimeragenesis. J Mol Biol 330:287–296
Farrow MF, Arnold FH (2010) Combinatorial recombination of gene fragments to construct a library of chimeras. Curr Protoc Protein Sci 62:26.2.1–26.2.20
Acknowledgments
The authors acknowledge funding from the Institute for Collaborative Biotechnologies through grant W911NF-09-D-0001 from the US Army Research Office and the National Central University, Taiwan, through a Cooperative Agreement for Energy Research Collaboration. M.A.S. is supported by a Resnick Sustainability Institute fellowship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Smith, M.A., Arnold, F.H. (2014). Designing Libraries of Chimeric Proteins Using SCHEMA Recombination and RASPP. In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1053-3_22
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-1052-6
Online ISBN: 978-1-4939-1053-3
eBook Packages: Springer Protocols