A universal, vector-based system for nucleic acid reading-frame selection
TL;DR: The development and experimental implementation of a general in-frame selection system, pSALect, a plasmid vector that utilizes two marker sequences flanking the DNA of interest that overcomes inconsistencies observed with traditional C-terminally fused reporter proteins.
read more
Abstract: The identification of a nucleic acid sequence's correct reading frame has important implications for homology-independent protein engineering techniques such as incremental truncation and SCRATCHY. We report the development and experimental implementation of a general in-frame selection system, pSALect, a plasmid vector that utilizes two marker sequences flanking the DNA of interest. This dual selection approach overcomes inconsistencies observed with traditional C-terminally fused reporter proteins. In the pSALect vector, sequences of interest are positioned between an N-terminal Tat-signal sequence and a C-terminal beta-lactamase reporter. In-frame selection of the resulting three-domain protein is performed by growing colonies on ampicillin-containing plates, requiring full-length translation in order to link covalently the signal sequence to the lactamase for export into the periplasm. This dual selection scheme has been validated successfully using defined sequences of glycinamide ribonucleotide formyltransferases (GARTs) from Escherichia coli and human and, in contrast to C-terminal fusion systems, proved effective when applied towards the selection of in-frame constructs in an incremental truncation library.
read more
Chat with Paper
AI Agents for this Paper
Find similar papers on Google Scholar, PubMed and Arxiv
Write a critical review of this paper
Analyze citations of this paper to find unaddressed research gaps
Citations
Novel methods for directed evolution of enzymes: quality, not quantity.
Stefan Lutz,Wayne M. Patrick +1 more
TL;DR: In the past decade methods of directed molecular evolution have proven revolutionary in protein engineering; an increasing number of powerful new combinatorial techniques have joined rational design methods as effective tools for the manipulation and tailoring of biocatalysts.
228
Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway
TL;DR: This work reports the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells and demonstrates that survival of Escherichia coli cells on selective medium expressing a Tat‐targeted test protein/β‐lactamase fusion correlates with the solubilities of the test protein.
152
Gene synthesis: Methods and applications
TL;DR: The techniques used in the generation of synthetic DNA are reviewed, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed.
91
Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity.
TL;DR: In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides using a β-lactamase selection strategy to maximize the functional diversity of the library.
78
Directed evolution of enzymes for biocatalysis and the life sciences.
TL;DR: Current enzyme engineers are still learning which combinations of selection methods and techniques for mutagenesis and DNA recombination are most efficient, and deciding where to introduce mutations or where to allow recombination is actively being investigated by combining experimental and computational methods.
71
References
Rapid protein-folding assay using green fluorescent protein.
TL;DR: This work demonstrated that the fluorescence of Escherichia coli cells expressing GFP fusions is related to the productive folding of the upstream protein domains expressed alone, providing a simple route to improving protein folding and expression by directed evolution.
991
Combinatorial protein engineering by incremental truncation
TL;DR: A combinatorial approach, using incremental truncation libraries of overlapping N- and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer, revealing that the active site could be bisected.
Genetic analysis of the twin arginine translocator secretion pathway in bacteria.
TL;DR: The development of a highly quantitative protein reporter for genetic analysis of Tat-specific export is reported, indicating that twin arginine translocation does not require the presence of anArginine dipeptide within the conserved sequence motif.
165
Detection of protein-protein interactions by protein fragment complementation strategies.
Stephen W. Michnick,Ingrid Remy,François-X. Campbell-Valois,Alexis Vallée-Bélisle,Joelle N. Pelletier +4 more
TL;DR: The generality of the PCA strategy is demonstrated with examples of assays that are designed on the basis of other enzymes including glycinamide ribonucleotide transformylase, aminoglycoside kinase, and hygromycin B kinase.
156
Processing of the psbA 5′ Untranslated Region in Chlamydomonas reinhardtii Depends upon Factors Mediating Ribosome Association
TL;DR: Results are consistent with a hierarchical maturation pathway for chloroplast messages, mediated by nuclear-encoded factors, that integrates mRNA processing, message stability, ribosome association, and translation.
63