Directed evolution is an exceptionally powerful tool that uses random mutant library generation and screening techniques to engineer or optimize functions of proteins. One class of proteins for which this process is particularly effective is antibodies, where properties such as antigen specificity and affinity can be selected to yield molecules with improved efficacy as molecular labels or in potential therapeutics. Typical antibody structure includes disulfide bonds that are required for stability and proper folding of the domains. However, these bonds are unable to form in the reducing environment of the cytoplasm, stymieing the effectiveness of optimized antibodies in many research applications. We have removed disulfide-forming cysteine residues in a single chain antibody fluorogen-activating protein (FAP), HL4, and employed directed evolution to select a derivative that is capable of activity in the cytoplasm. A subsequent round of directed evolution was targeted at increasing the overall brightness of the fluoromodule (FAP-fluorogen complex). Ultimately, this approach produced a novel FAP that exhibits strong activation of its cognate fluorogen in the reducing environment of the cytoplasm, significantly expanding the range of applications for which fluoromodule technology can be utilized.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633666 | PMC |
| http://dx.doi.org/10.1007/s12033-012-9631-7 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
"Chance and Necessity" on the Molecular Evolution of REV3 (a Catalytic Subunit of DNA Polymerase ζ)-The Dual Roles of Translesion and Neuronal Extension.
Genes Cells
January 2025
Department of Genetic Biochemistry, The National Institutes of Biomedical Innovation, Health and Nutrition, Shinjuku-ku, Tokyo, Japan.
Catalytic subunit of DNA polymerase ζ (REV3), involved in translesion-replication is evolutionarily conserved from yeast and plants to higher eukaryotes. However, a large intermediate domain is inserted in REV3 of humans and mice. The domain has "DUF4683" region, which is significantly similar to human neurite extension and migration factor (NEXMIF).
View Article and Find Full Text PDFOptimized directed evolution of E. coli leucyl-tRNA synthetase adds many noncanonical amino acids into the eukaryotic genetic code including ornithine and Nε-acetyl-methyllysine.
Angew Chem Int Ed Engl
January 2025
Boston College, Chemistry, 2609 Beacon Street, 201 Merkert Chemistry Center, 02467, Chestnut hill, UNITED STATES OF AMERICA.
Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in eukaryotes has predominantly relied on the pyrrolysyl-tRNA synthetase/tRNA pair. However, access to additional easily engineered pairs is crucial for expanding the structural diversity of the ncAA toolbox in eukaryotes. The Escherichia coli-derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair presents a particularly promising alternative.
View Article and Find Full Text PDFDirect Evidence of Microbial Sunscreen Production by Scum-Forming Cyanobacteria in the Baltic Sea.
Environ Microbiol Rep
February 2025
Department of Microbiology, University of Helsinki, Helsinki, Finland.
Mycosporine-like amino acids are water-soluble secondary metabolites that protect photosynthetic microorganisms from ultraviolet radiation. Here, we present direct evidence for the production of these compounds in surface scums of cyanobacteria along the Baltic Sea coast. We collected 59 environmental samples from the southern coast of Finland during the summers of 2021 and 2022 and analysed them using high-resolution liquid chromatography-mass spectrometry.
View Article and Find Full Text PDFThe Landmark Series: Testing in Thyroid Nodule Fine Needle Aspiration Cytology.
Ann Surg Oncol
January 2025
University of Pittsburgh, Pittsburgh, PA, USA.
There has been remarkable growth in our understanding of the biologic behavior and molecular signature of thyroid malignancies, which has led to the introduction, application, and evolution of molecular testing of thyroid nodule FNA cytology. Next-generation sequencing molecular testing is an important, well-validated diagnostic tool for management of cytologically indeterminate thyroid nodules. It reduces unnecessary thyroid surgery for benign disease, avoiding associated surgical risks for the patient and reducing healthcare expenses.
View Article and Find Full Text PDFEvolution and Functional Diversification of Serine Racemase Homologs in Bacteria.
J Mol Evol
January 2025
Department of Plant and Soil Sciences, 311 Plant Science Building, University of Kentucky, Lexington, KY, 40546-0312, USA.
Amino acid racemases catalyze the interconversion of L- and D-amino acids, maintaining intracellular levels of both D- and L-amino acids. While alanine and glutamate racemases are widespread in bacteria, serine racemase (SerR) is predominantly found in animals. Recently, homologs of animal SerR were reported in some bacterial genomes, but their evolutionary distribution and functional roles remain poorly understood.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!