Directed Evolution of Proteins for Device Applications

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

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Directed Evolution of Proteins throughIn VitroProtein Synthesis in Liposomes

Journal of Nucleic Acids ◽

10.1155/2012/923214 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 12

Author(s):

Takehiro Nishikawa ◽

Takeshi Sunami ◽

Tomoaki Matsuura ◽

Tetsuya Yomo

Keyword(s):

Protein Synthesis ◽

Directed Evolution ◽

Single Copy ◽

Functional Evaluation ◽

Future Directions ◽

Pure System ◽

Protein Functions ◽

A Cell ◽

Evolution Of Proteins

Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.”In vitrocompartmentalization (IVC) is anin vitrogene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment,in vitroprotein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized byin vitroprotein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP andβ-glucuronidase are described. We discuss the future directions for this method and its applications.

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AXM mutagenesis: An efficient means for the production of libraries for directed evolution of proteins

Journal of Immunological Methods ◽

10.1016/j.jim.2013.05.003 ◽

2013 ◽

Vol 394 (1-2) ◽

pp. 55-61 ◽

Cited By ~ 8

Author(s):

Erika G. Holland ◽

Diane L. Buhr ◽

Felicity E. Acca ◽

Dawn Alderman ◽

Kristin Bovat ◽

...

Keyword(s):

Directed Evolution ◽

Evolution Of Proteins

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TRIAD: a transposition-based approach for gene mutagenesis by random short in-frame insertions and deletions for directed protein evolution

10.21203/rs.3.pex-1448/v1 ◽

2021 ◽

Author(s):

Stephane Emond ◽

Florian Hollfelder

Keyword(s):

Directed Evolution ◽

Protein Evolution ◽

Transposon Mutagenesis ◽

Scfv Antibody ◽

Standard Protocol ◽

Insertions And Deletions ◽

Natural Protein ◽

Nucleotide Insertions ◽

Evolution Of Proteins

Abstract Insertions and deletions (InDels) are among the most frequent changes observed in natural protein evolution, yet their potential has hardly been harnessed in directed evolution experiments. Here we describe the standard protocol for TRIAD (Transposition-based Random Insertion And Deletion mutagenesis), a simple and efficient Mu transposon mutagenesis approach for generating libraries of single InDel variants with one, two or three triplet nucleotide insertions or deletions. This method has recently been employed in three published examples of InDel-based directed evolution of proteins, including a phosphotriesterase, a scFv antibody and an ancestral luciferase.

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