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| Open AccessA cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies
Faster, higher throughput antibody engineering methods are needed. Here the authors present CeVICA, a cell-free nanobody engineering platform using ribosome display and computational clustering analysis for in vitro selection; they use this to develop nanobodies against the RBD of SARS-CoV-2 spike protein.
- Xun Chen
- , Matteo Gentili
- & Aviv Regev
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Article
| Open AccessOne-step construction of circularized nanodiscs using SpyCatcher-SpyTag
Circularised nanodiscs (cNDs) are able to stabilise large lipid bilayer patches and are used for structural and functional studies. Current techniques to build cNDs have numerous steps and low yields; here the authors report a single step construction method using the SpyCatcher-SpyTag system.
- Shanwen Zhang
- , Qian Ren
- & Huan Bao
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Article
| Open AccessRole of backbone strain in de novo design of complex α/β protein structures
The authors show that consideration of global backbone strain enables successful de novo design of larger αβ-proteins with five- and six- stranded β-sheets flanked by α-helices.
- Nobuyasu Koga
- , Rie Koga
- & David Baker
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Article
| Open AccessSingle-component near-infrared optogenetic systems for gene transcription regulation
Current near-IR optogenetic systems to regulate transcription consist of a number of large protein components. Here the authors report a smaller single-component near-IR system, iLight, developed from a bacterial phytochrome that they use to control gene transcription in bacterial and mammalian cells.
- Andrii A. Kaberniuk
- , Mikhail Baloban
- & Vladislav V. Verkhusha
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Article
| Open AccessEngineering an anti-HER2 biparatopic antibody with a multimodal mechanism of action
HER2 acts an oncogenic driver in numerous cancers. Here, the authors design an anti-HER2 biparatopic and tetravalent IgG fusion with inhibitory effects in a xenograft model.
- Florian Kast
- , Martin Schwill
- & Andreas Plückthun
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Article
| Open Accessα-Helical peptidic scaffolds to target α-synuclein toxic species with nanomolar affinity
α-Synuclein (αS) aggregation is a driver of several neurodegenerative disorders. Here, the authors identify a class of peptides that bind toxic αS oligomers and amyloid fibrils but not monomeric functional protein, and prevent further αS aggregation and associated cell damage.
- Jaime Santos
- , Pablo Gracia
- & Salvador Ventura
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Article
| Open AccessEngineering the protein dynamics of an ancestral luciferase
Directed evolution commonly relies on point mutations but InDels frequently occur in evolution. Here the authors report a protein-engineering framework based on InDel mutagenesis and fragment transplantation resulting in greater catalysis and longer glow-type bioluminescence of the ancestral luciferase.
- Andrea Schenkmayerova
- , Gaspar P. Pinto
- & Jiri Damborsky
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Article
| Open AccessIntracellular artificial supramolecules based on de novo designed Y15 peptides
Self-assembling peptides (SAPs) can be used to build biomaterials, but genetically encoded SAPs have rarely been used as building blocks in cells. Here, the authors design a SAP that can be genetically fused to target proteins to induce their intracellular clustering and modulate their signaling functions.
- Takayuki Miki
- , Taichi Nakai
- & Hisakazu Mihara
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Article
| Open AccessAnchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites
Cyclic peptides are of particular interest due to their pharmacological properties, but their design for binding to a target protein is challenging. Here, the authors present a computational “anchor extension” methodology for de novo design of cyclic peptides that bind to the target protein with high affinity, and validate the approach by developing cyclic peptides that inhibit histone deacetylases 2 and 6.
- Parisa Hosseinzadeh
- , Paris R. Watson
- & David Baker
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Article
| Open AccessProtein design and variant prediction using autoregressive generative models
The ability to design functional sequences is central to protein engineering and biotherapeutics. Here the authors introduce a deep generative alignment-free model for sequence design applied to highly variable regions and design and test a diverse nanobody library with improved properties for selection experiments.
- Jung-Eun Shin
- , Adam J. Riesselman
- & Debora S. Marks
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Article
| Open AccessDesign of multi-scale protein complexes by hierarchical building block fusion
De novo design of self-assembling protein nanostructures and materials is of significant interest, however design of complex, multi-component assemblies is challenging. Here, the authors present a stepwise hierarchical approach to build such assemblies using helical repeat and helical bundle proteins as building blocks, and provide an in-depth structural characterization of the resulting assemblies.
- Yang Hsia
- , Rubul Mout
- & David Baker
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Article
| Open AccessDesign of buried charged networks in artificial proteins
Buried charged networks in proteins are often important for their biological functionality and are believed to destabilise the protein fold. Here, the authors combine computational design, MD simulations, biophysical experiments, NMR and X-ray crystallography to design and characterise artificial 4α-helical proteins with buried charged elements. They analyse their conformational landscapes and observe that the ion-pairs are stabilised by amphiphilic residues that electrostatically shield the charged motif, which increases structural stability.
- Mona Baumgart
- , Michael Röpke
- & Ville R. I. Kaila
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Article
| Open AccessHinge-shift mechanism as a protein design principle for the evolution of β-lactamases from substrate promiscuity to specificity
TEM-1 β-lactamase evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency and degrades β-lactam antibiotics with a strong preference for penicillins. Here authors developed a computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism and show a putative Precambrian β-lactamase that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements.
- Tushar Modi
- , Valeria A. Risso
- & S. Banu Ozkan
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| Open AccessStructural resolution of switchable states of a de novo peptide assembly
So far most of the de novo designed proteins are for single states only. Here, the authors present the de novo design and crystal structure determination of a coiled-coil peptide that assembles into multiple, distinct conformational states under the same conditions and further characterise its properties with biophysical experiments, NMR and MD simulations.
- William M. Dawson
- , Eric J. M. Lang
- & Derek N. Woolfson
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Article
| Open AccessLasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins
RaPID (Random non-standard Peptides Integrated Discovery) enables discovery of small macrocyclic peptides binding desired targets. Here, the authors propose lasso-grafting: the RaPID-derived peptides are implanted onto diverse proteins and maintain both the binding properties of the cyclic peptide and the host protein function.
- Emiko Mihara
- , Satoshi Watanabe
- & Junichi Takagi
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Article
| Open AccessDe novo design of a reversible phosphorylation-dependent switch for membrane targeting
The ability to dynamically control protein-protein interactions and localization of proteins is critical in synthetic biological systems. Here the authors develop a peptide-based molecular switch that regulates dimer formation and lipid membrane targeting via reversible phosphorylation.
- Leon Harrington
- , Jordan M. Fletcher
- & Petra Schwille
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Article
| Open AccessActive-site loop variations adjust activity and selectivity of the cumene dioxygenase
Active-site loops are important for catalytic properties of enzymes, but challenging to engineer due to their high flexibility and diversity. Here, the authors identify and engineer hot-spots in the loops of cumene dioxygenase, obtain variants with changed activity, regio- and enantioselectivity, and present a Linker In Loop Insertion approach for loop modification.
- Peter M. Heinemann
- , Daniel Armbruster
- & Bernhard Hauer
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Article
| Open AccessSelf-assembly and regulation of protein cages from pre-organised coiled-coil modules
Coiled-coil protein origami is a strategy for the de novo design of polypeptide nanostructures based on coiled-coil dimer forming peptides, where a single chain protein folds into a polyhedral cage. Here, the authors design a single-chain triangular bipyramid and also demonstrate that the bipyramid can be self-assembled as a heterodimeric complex, comprising pre-defined subunits.
- Fabio Lapenta
- , Jana Aupič
- & Roman Jerala
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Article
| Open AccessDesigned folding pathway of modular coiled-coil-based proteins
Coiled-coil protein origami (CCPO) is a strategy for the design of polyhedral cage-shaped protein folds based on coiled-coil (CC) dimer-forming peptides. Here, the authors show that CCPO proteins fold in a multistep process governed by the spatial distance between CC modules in the primary sequence and subsequent folding intermediates, which enables the use of identical CC modules for the CCPO tetrahedron design.
- Jana Aupič
- , Žiga Strmšek
- & Roman Jerala
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Article
| Open AccessIncorporation of sensing modalities into de novo designed fluorescence-activating proteins
Fluorescent protein reporters based on GFP exist, but have intrinsic disadvantages. Here the authors incorporate pH, Ca2+ and protein–protein interaction sensing modalities into de novo designed mini-fluorescence-activating proteins (mFAPs), with increased photostability and smaller size, which bind a range of DFHBI chromophore variants.
- Jason C. Klima
- , Lindsey A. Doyle
- & David Baker
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Article
| Open AccessDirect control of CAR T cells through small molecule-regulated antibodies
Many next-generation antibody therapeutics have enhanced potency but the risk of adverse events. Here the authors develop a conditionally activated, single-module CAR.
- Spencer Park
- , Edward Pascua
- & Javier Chaparro-Riggers
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Article
| Open AccessNeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins
Covalent conjugation of endogenous protein complexes offers many opportunities for fundamental and clinical research. Based on a bacterial protein domain that forms a reactive anhydride in the presence of Ca2+, the authors here develop a system that enables the covalent capture of endogenous binding partners.
- Arne H. A. Scheu
- , Sheryl Y. T. Lim
- & Mark Howarth
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Article
| Open AccessHeme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase
Family 1 glycosidases (GH1) are present in the three domains of life and share classical TIM-barrel fold. Structural and biochemical analyses of a resurrected ancestral GH1 enzyme reveal heme binding, not known in its modern descendants. Heme rigidifies the TIM-barrel and allosterically enhances catalysis.
- Gloria Gamiz-Arco
- , Luis I. Gutierrez-Rus
- & Jose M. Sanchez-Ruiz
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Article
| Open AccessEngineering of a bona fide light-operated calcium channel
Existing optogenetic methods to induce calcium mobilisation lack selectivity and specificity. Here, the authors design and engineer a single-component light-operated calcium channel to provide optical control over calcium signals and calcium-dependent physiological responses: LOCa.
- Lian He
- , Liuqing Wang
- & Yubin Zhou
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Article
| Open AccessA nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure
Characterizing the assembly of the nuclear pore complex (NPC) remains challenging. Here, the authors develop a set of nanobodies that recognize seven constituent nucleoporins, study their binding characteristics, and apply them to probe accessible and obstructed NPC surfaces in yeast.
- Sarah A. Nordeen
- , Kasper R. Andersen
- & Thomas U. Schwartz
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Article
| Open AccessScalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities
Generating rich ortholog diversity for biocatalysts can be difficult due to the deep evolutionary processes involved. Here the authors use OrthoRep to rapidly evolve TrpB to produce sequence-diverse variants with altered substrate promiscuity.
- Gordon Rix
- , Ella J. Watkins-Dulaney
- & Chang C. Liu
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Article
| Open AccessGamma estimator of Jarzynski equality for recovering binding energies from noisy dynamic data sets
Measuring interaction energies from experimentally measured single-molecular interactions is challenging. Here, the authors report a gamma work distribution applied to single molecule pulling events for estimating peptide absorption free energy.
- Zhifeng Kuang
- , Kristi M. Singh
- & Rajesh R. Naik
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Article
| Open AccessTwo-tier supramolecular encapsulation of small molecules in a protein cage
Protein cages are used for encapsulation of macromolecules such as proteins and nucleic acids. Here, the authors report a strategy to encapsulate poorly soluble small molecules within porous protein cages through capsid-templated micelle formation and show that the resulting lipoprotein-like cages enhance the cellular delivery of these molecules.
- Thomas G. W. Edwardson
- , Stephan Tetter
- & Donald Hilvert
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Article
| Open AccessEngineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins
ADP-ribose binding macro domains facilitate the enrichment and detection of cellular ADP-ribosylation. Here, the authors generate an engineered macro domain with increased ADP-ribose affinity, improving the identification of ADP-ribosylated proteins by proteomics, western blot and immunofluorescence.
- Kathrin Nowak
- , Florian Rosenthal
- & Michael O. Hottiger
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Article
| Open AccessResonance energy transfer sensitises and monitors in situ switching of LOV2-based optogenetic actuators
Cellular optogenetics applications are limited by difficulties in quantification and blue light toxicity. Here the authors design LOV2-based switches that use resonance energy transfer to overcome these concerns.
- Li-Li Li
- , Florence M. Klein
- & Michael J. Courtney
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Article
| Open AccessA versatile soluble siglec scaffold for sensitive and quantitative detection of glycan ligands
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunomodulatory receptors expressed on cells of the hematopoietic lineage. Here the authors demonstrate an approach for the identification of the glycan ligands of Siglecs, which is also applicable to other families of glycan-binding proteins.
- Emily Rodrigues
- , Jaesoo Jung
- & Matthew S. Macauley
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Article
| Open AccessEnsemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico
Kemp eliminases are artificial enzymes that catalyze the concerted deprotonation and ring-opening of benzisoxazoles. Here, the authors use room-temperature X-ray crystallography to investigate changes to the conformational ensemble of the Kemp eliminase HG3 along a directed evolutionary trajectory, and develop an experimentally guided, ensemble-based computational enzyme design procedure.
- Aron Broom
- , Rojo V. Rakotoharisoa
- & Roberto A. Chica
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Article
| Open AccessModulating mechanical stability of heterodimerization between engineered orthogonal helical domains
Mechanically stable specific heterodimerization formed with reversible bonds are used as a molecular anchorage in single-molecule force spectroscopy studies with unique mechanical properties. Here authors develop a variety of heterodimerization molecular systems with a range of mechanical stability from a set of recently engineered helix-heterotetramers.
- Miao Yu
- , Zhihai Zhao
- & Jie Yan
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Article
| Open AccessEngineered systems of inducible anti-repressors for the next generation of biological programming
Transcriptional anti-repressors have been largely absent in the design of regulated genetic circuits. Here, the authors present a workflow of the engineering of non-natural anti-reperssors that can be built into NOT oriented logic gates.
- Thomas M. Groseclose
- , Ronald E. Rondon
- & Corey J. Wilson
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| Open AccessRibosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro
Backbone extended monomers are poorly compatible with the natural ribosomes, impeding their polymerization into polypeptides. Here the authors design non-canonical amino acid analogs with cyclic structures or extended carbon chains and used an engineered ribosome to improve tRNA-charging and incorporation into peptides.
- Joongoo Lee
- , Kevin J. Schwarz
- & Michael C. Jewett
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| Open AccessDevelopment of light-responsive protein binding in the monobody non-immunoglobulin scaffold
The ability to reversibly control monobody binding affinity would find use in biotechnology and research applications. Here the authors fuse the light-sensitive AsLOV2 domain to a monobody against the Abl SH2 domain to obtain a light dependent monobody and apply it in vitro and in mammalian cells.
- César Carrasco-López
- , Evan M. Zhao
- & José L. Avalos
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Article
| Open AccessA general approach to engineer positive-going eFRET voltage indicators
Genetically encoded voltage indicators (GEVIs) allow visualisation of fast action potentials in neurons but most are bright at rest and dimmer during an action potential. Here, the authors engineer electrochromic FRET GEVIs with fast, bright and positive-going fluorescence signals for in vivo imaging.
- Ahmed S. Abdelfattah
- , Rosario Valenti
- & Eric R. Schreiter
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Article
| Open AccessAccessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis
Insertions/Deletions (InDels) remain an untapped source of protein diversity in laboratory evolution. The method TRIAD generates libraries of random variants with short in-frame InDels using transposons, allowing a comparison of their evolutionary potential with widely-used point mutant libraries.
- Stephane Emond
- , Maya Petek
- & Florian Hollfelder
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Article
| Open AccessMolecular basis for the distinct functions of redox-active and FeS-transfering glutaredoxins
Glutaredoxins are a family of essential enzymes divided into two major classes with either a CGFS or a CxxC active site, of which only the latter exhibits oxidoreductase activity. Here the authors address the structural basis for the functional difference between the two classes of glutaredoxins.
- Daniel Trnka
- , Anna D. Engelke
- & Christopher Horst Lillig
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Article
| Open AccessChimeric design of pyrrolysyl-tRNA synthetase/tRNA pairs and canonical synthetase/tRNA pairs for genetic code expansion
Orthogonal aminoacyl-tRNA synthetase/tRNA pairs are crucial for the incorporation of unnatural amino acids in a site-specific manner. Here the authors use rational chimera design to create multiple efficient pairs that function in bacterial and mammalian systems for genetic code expansion.
- Wenlong Ding
- , Hongxia Zhao
- & Shixian Lin
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Article
| Open AccessEnhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase
Malate dehydrogenase (MDH) is one of the key enzymes for succinic acid (SA) bioproduction. Here, the authors report biochemical and structural analyses of various MDHs to reveal amino acids influencing the specific activity and susceptibility to substrate inhibition, and achieve industrial-level SA production.
- Jung Ho Ahn
- , Hogyun Seo
- & Sang Yup Lee
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Article
| Open AccessPolychromatic solar energy conversion in pigment-protein chimeras that unite the two kingdoms of (bacterio)chlorophyll-based photosynthesis
The spectra of light used by photosynthetic organisms are determined by their pigmentation colour palettes. Here Liu et al. show that a genetically-encoded chimera of light-harvesting proteins from plants and reaction centres from purple bacteria allows for polychromatic solar energy harvesting.
- Juntai Liu
- , Vincent M. Friebe
- & Michael R. Jones
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Article
| Open AccessStructures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection
Major Histocompatibility Complex (MHC) class I molecules present tightly binding peptides on the cell surface for recognition by cytotoxic T cells. Here, the authors present the crystal structures of a disulfide-stabilized human MHC class I molecule in the peptide-free state and bound with dipeptides, and find that peptide binding is accompanied by concerted conformational switches of the amino acid side chains in the binding pockets.
- Raghavendra Anjanappa
- , Maria Garcia-Alai
- & Rob Meijers
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Article
| Open AccessBlackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs
Mutations to SpCas9 can improve fidelity and mitigate off-target effects. Here the authors generate ‘Blackjack’ mutations that improve fidelity while retaining effectiveness with 21G-sgRNAs.
- Péter István Kulcsár
- , András Tálas
- & Ervin Welker
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Article
| Open AccessA toxin-antidote CRISPR gene drive system for regional population modification
CRISPR homing gene drives are highly invasive and can fail due to the rapid evolution of resistance. Here the authors present TARE drive, inspired by naturally occurring selfish genetic elements, which is less vulnerable to resistance and can potentially be confined to a target population.
- Jackson Champer
- , Esther Lee
- & Philipp W. Messer
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Article
| Open AccessCysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones
Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Here the authors report 3-bromo-5-methylene pyrrolones as a simple, robust and versatile class of reagents for cysteine-specific protein modification.
- Yingqian Zhang
- , Chuanlong Zang
- & Chuanzheng Zhou
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Article
| Open AccessMinimally disruptive optical control of protein tyrosine phosphatase 1B
Protein tyrosine phosphatases regulate many cellular processes but are difficult to study in their native context. Here the authors develop an approach for using light to control the activity of a disease-relevant phosphatase without interfering with its native cellular organization.
- Akarawin Hongdusit
- , Peter H. Zwart
- & Jerome M. Fox
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Article
| Open AccessAn ultra-stable cytoplasmic antibody engineered for in vivo applications
Antibodies expressed in the cytosol often form insoluble aggregates, which makes it hard to target intracellular proteins. Here the authors engineer an ultra-stable cytoplasmic antibody (STAND) with a low isoelectric point that can be used in vivo.
- Hiroyuki Kabayama
- , Makoto Takeuchi
- & Katsuhiko Mikoshiba
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Article
| Open AccessA set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales
Monomeric near-infrared (NIR) fluorescent proteins (FPs) from bacterial phytochromes bring potential advantages, but their brightness in cells is lower than dimeric NIR FPs. Here the authors develop enhanced monomeric NIR FPs enabling imaging across different scales without the trade-off between brightness and monomeric state.
- Mikhail E. Matlashov
- , Daria M. Shcherbakova
- & Vladislav V. Verkhusha