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

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.

scholarly journals Advances in the directed evolution of proteins

2014 ◽  
Vol 22 ◽  
pp. 129-136 ◽  
Author(s):  
Michael D Lane ◽  
Burckhard Seelig
Keyword(s):  
Directed Evolution ◽  
Evolution Of Proteins

Directed Evolution of Proteins for Device Applications

2008 ◽  
Author(s):  
Jeremy Koscielecki ◽  
Jason Hillebrecht ◽  
Robert Birge
Keyword(s):  
Directed Evolution ◽  
Device Applications ◽  
Evolution Of Proteins

scholarly journals Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Benjamin O. Fulton ◽  
David Sachs ◽  
Megan C. Schwarz ◽  
Peter Palese ◽  
Matthew J. Evans
Keyword(s):  
Zika Virus ◽  
Viral Evolution ◽  
Rna Replication ◽  
Transposon Mutagenesis ◽  
Ns1 Protein ◽  
Genetic Constraints ◽  
E Protein ◽  
A Genome ◽  
Nucleotide Insertions ◽  
Fusion Loop

ABSTRACT The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus. IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses.

scholarly journals Laboratory-Directed Protein Evolution

2005 ◽  
Vol 69 (3) ◽  
pp. 373-392 ◽  
Author(s):  
Ling Yuan ◽  
Itzhak Kurek ◽  
James English ◽  
Robert Keenan
Keyword(s):  
Protein Evolution ◽  
Selective Pressure ◽  
Evolutionary Potential ◽  
Effective Strategies ◽  
Scientific Disciplines ◽  
Wide Range ◽  
Protein Functions ◽  
Evolution Of Proteins ◽  
Efficient Screening ◽  
New Protein

SUMMARY Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences.

scholarly journals Protein evolution with an expanded genetic code

2008 ◽  
Vol 105 (46) ◽  
pp. 17688-17693 ◽  
Author(s):  
Chang C. Liu ◽  
Antha V. Mack ◽  
Meng-Lin Tsao ◽  
Jeremy H. Mills ◽  
Hyun Soo Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Unnatural Amino Acids ◽  
Selective Advantage ◽  
Display System ◽  
Antibody Library ◽  
Evolution Of Proteins ◽  
Expanded Genetic Code ◽  
Selection Of

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.

scholarly journals Directed Evolution of Proteins throughIn VitroProtein Synthesis in Liposomes

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
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.

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

2013 ◽  
Vol 394 (1-2) ◽  
pp. 55-61 ◽  
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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