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| Open AccessDiverse protein assembly driven by metal and chelating amino acids with selectivity and tunability
Precise manipulation of protein self-assembly in vitro is challenging. Here, the authors developed an approach for driving metal-mediated reversible protein assembly by genetically installing a bipyridine residue into an oligomeric (D3) protein.
- Minwoo Yang
- & Woon Ju Song
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Article
| Open AccessAn engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence
Lee et al. report an engineered IgG1 Fc domain that behaves like an hFcRn binding pH toggle switch. The authors show that this new half-life extension Fc domain confers improved pharmacokinetics in new humanized knock-in mouse strains that recapitulate the key processes for antibody persistence in circulation.
- Chang-Han Lee
- , Tae Hyun Kang
- & George Georgiou
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Article
| Open AccessEvaluation of integrin αvβ6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis
Knottin is a cystine knot peptide. Here, the authors develop a knottin-based tracer for positron emission tomography and demonstrate its ability to detect cancer and idiopathic pulmonary fibrosis through selective binding to integrin αvβ6.
- Richard H. Kimura
- , Ling Wang
- & Sanjiv S. Gambhir
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Article
| Open AccessEngineering selective competitors for the discrimination of highly conserved protein-protein interaction modules
Developing inhibitors that target specific protein-protein interactions (PPIs) is challenging. Here, the authors show that target selectivity and PPI blocking can be achieved simultaneously with PPI inhibitors that contain two functional modules, and create a paralog-selective PSD-95 inhibitor as proof-of-concept.
- Charlotte Rimbault
- , Kashyap Maruthi
- & Matthieu Sainlos
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Article
| Open AccessFramework engineering to produce dominant T cell receptors with enhanced antigen-specific function
Increasing TCR cell surface expression can potentiate T cell responses to low-concentrations of antigen. Here the authors identify aminoacids in human TCR variable domains that impact its surface expression, and demonstrate how editing these residues can improve T cell activation and effector function without altering antigen specificity.
- Sharyn Thomas
- , Fiyaz Mohammed
- & Hans J. Stauss
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Article
| Open AccessHuman immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility
Conserved regions of the antibody molecule impact its downstream biological effects. Here the authors show that a rigid hinge conformation increases the agonistic activity of CD40 and DR5 antibodies, distinctly from FcγR-binding, suggesting that the hinge and FcR binding regions can be separately modified to optimize therapies.
- Xiaobo Liu
- , Yingjie Zhao
- & Fubin Li
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Article
| Open AccessEnhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle
Nanoparticles are a promising approach to increase immunogenicity of protein antigens for vaccines. Here, Brouwer et al. design self-assembling, two-component protein NPs that present native-like SOSIP trimers of HIV envelope protein and determine immunogenicity in a small animal model.
- Philip J. M. Brouwer
- , Aleksandar Antanasijevic
- & Rogier W. Sanders
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Article
| Open AccessArtificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution
Candida antarctica lipase B (CALB) is a serine lipase. Here, the authors use directed evolution to exchange serine with cysteine in the catalytic triad of the enzyme, thereby obtaining a highly active CALB variant that — unlike the wild type — accommodates bulky substrates.
- Yixin Cen
- , Warispreet Singh
- & Manfred T. Reetz
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Article
| Open AccessArtificial coiled coil biomineralisation protein for the synthesis of magnetic nanoparticles
Proteins have been used in the synthesis of magnetic nanoparticles but issues with aggregation limit this application. Here, the authors report on the synthesis of coiled proteins that display the active loop of the natural proteins to avoid aggregation and investigate the application in nanoparticle synthesis.
- Andrea E. Rawlings
- , Lori A. Somner
- & Sarah S. Staniland
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Article
| Open AccessSide chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor
Polyglutamine (polyQ) tracts are low-complexity regions and their expansion is linked to certain neurodegenerative diseases. Here the authors combine experimental and computational approaches to find that the length of the androgen receptor polyQ tract correlates with its helicity and show that the polyQ helical structure is stabilized by hydrogen bonds between the Gln side chains and main chain carbonyl groups.
- Albert Escobedo
- , Busra Topal
- & Xavier Salvatella
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Article
| Open AccessA 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold
Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo‐electron microscopy (cryo‐EM). Here authors design a protein scaffold that binds 12 copies of a small 26 kDa protein (GFP), which allowed visualizing GFP at a resolution of 3.8Å by cryo‐EM.
- Yuxi Liu
- , Duc T. Huynh
- & Todd O. Yeates
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Article
| Open AccessA survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins
Protein phosphorylation helps to control many important cellular activities. Here the authors describe a genetic selection strategy to isolate designed ankyrin repeat proteins that bind specifically to phosphomodified targets.
- Bunyarit Meksiriporn
- , Morgan B. Ludwicki
- & Matthew P. DeLisa
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Article
| Open AccessDiversifying the structure of zinc finger nucleases for high-precision genome editing
Genome editing often requires cleavage within a narrow sequence window. Here the authors develop an expanded set of zinc finger nuclease architectures that increase the available configurations by a factor of 64 and can target almost every base at loci of therapeutic significance.
- David E. Paschon
- , Stephanie Lussier
- & Edward J. Rebar
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Article
| Open AccessNeurotrophin receptor tyrosine kinases regulated with near-infrared light
Optical control over receptor tyrosine kinase (RTK) activity can find application in both basic biology and synthetic biology. Here the authors combine the photosensory module of DrBphP bacterial phytochrome with the Trk family of RTKs to obtain Trks that are reversibly switchable with near-infrared and far-red light.
- Anna V. Leopold
- , Konstantin G. Chernov
- & Vladislav V. Verkhusha
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Article
| Open AccessPromiscuous enzymatic activity-aided multiple-pathway network design for metabolic flux rearrangement in hydroxytyrosol biosynthesis
Metabolic engineering usually focuses on manipulating enzyme(s) within a single pathway. Here, the authors show that a promiscuous enzymatic activity-based multiple-pathway design can minimize cell metabolic burden and allow carbon flow rearrangement, leading to efficient hydroxytyrosol biosynthesis.
- Wei Chen
- , Jun Yao
- & Shuang-Yan Tang
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Article
| Open AccessRationally designed carbohydrate-occluded epitopes elicit HIV-1 Env-specific antibodies
Areas of HIV envelope (Env) that aren’t covered by glycans are potential targets for antibodies. Here, the authors computationally design small protein mimics of four such epitopes and show that they can induce Env binding antibodies in rabbits.
- Cheng Zhu
- , Elena Dukhovlinova
- & Nikolay V. Dokholyan
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Article
| Open AccessDisulfide-mediated conversion of 8-mer bowl-like protein architecture into three different nanocages
Shape transformation of proteins created by design in the laboratory is challenging. Here, the authors present a disulfide-mediated approach for the preparation of 16-mer, 24-mer, and 48-mer nanocages from an 8-mer bowl-like protein building block.
- Jiachen Zang
- , Hai Chen
- & Guanghua Zhao
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| Open AccessEngineering of CRISPR-Cas12b for human genome editing
The Cas12b family of CRISPR nucleases has been underutilized in mammalian cells due to the high temperature requirement of known members. Here the authors engineer BhCas12b to overcome this limitation for robust and specific genome editing applications in human cells.
- Jonathan Strecker
- , Sara Jones
- & Feng Zhang
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Article
| Open AccessSmallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing
Near-infrared (NIR) fluorescent proteins (FPs) offer advantages for spectral multiplexing and deep-tissue imaging. Here the authors engineer a smaller NIR FP based on the unexplored cyanobacteriochrome photoreceptor and demonstrate its use in various applications in cell culture as well as whole-body imaging in vivo in mice.
- Olena S. Oliinyk
- , Anton A. Shemetov
- & Vladislav V. Verkhusha
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Article
| Open AccessLocally anchoring enzymes to tissues via extracellular glycan recognition
The use of enzymes as drugs requires that they persist within target tissues over therapeutically relevant time frames. Here the authors use a galectin-3 fusion to anchor enzymes at injection sites for up to 14 days.
- Shaheen A. Farhadi
- , Evelyn Bracho-Sanchez
- & Gregory A. Hudalla
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Article
| Open AccessShort-chain ketone production by engineered polyketide synthases in Streptomyces albus
Mutating natural enzymes is effective in broadening the substrate or product range, but generally leads to reduced titers. Here the authors engineer hybrid polyketide synthases for efficient production of short-chain ketones from plant biomass hydrolysates in Streptomyces, which can increase the octane of gasoline.
- Satoshi Yuzawa
- , Mona Mirsiaghi
- & Jay D. Keasling
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Article
| Open AccessImproved methods for marking active neuron populations
Methods to directly label active neurons are still lacking. Here the authors develop CaMPARI2, a photoconvertible fluorescent protein sensor for neuronal activity with improved brightness and calcium binding kinetics, as well as an antibody to amplify the activated sensor signal in fixed samples.
- Benjamien Moeyaert
- , Graham Holt
- & Eric R. Schreiter
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Article
| Open AccessMaintaining and breaking symmetry in homomeric coiled-coil assemblies
Higher order coiled coils with five or more helices can form α-helical barrels. Here the authors show that placing β-branched aliphatic residues along the lumen yields stable and open α-helical barrels, which is of interest for the rational design of functional proteins; whereas, the absence of β-branched side chains leads to unusual low-symmetry α-helical bundles.
- Guto G. Rhys
- , Christopher W. Wood
- & Derek N. Woolfson
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Article
| Open AccessComputational design of chemogenetic and optogenetic split proteins
Designing split protein approaches is time consuming and often results in high background activity due to spontaneous assembly. Here the authors present an automated approach which uses a split energy scoring function to identify optimal protein split sites and reduces spontaneous assembly.
- Onur Dagliyan
- , Andrey Krokhotin
- & Nikolay V. Dokholyan
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Article
| Open AccessMapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries
Characterizing the binding selectivity landscape of interacting proteins is crucial in protein engineering. Here the authors use multi-target selective library screening and in silico next-generation sequencing to map the binding landscape of proteins and produce improved proteases inhibitors.
- Si Naftaly
- , Itay Cohen
- & Niv Papo
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Article
| Open AccessPoreDesigner for tuning solute selectivity in a robust and highly permeable outer membrane pore
Monodisperse angstrom-sized membrane proteins, found in biological membranes, are difficult to implement in synthetic industrial membranes. Here authors present a pore design procedure, to redesign the robust beta-barrel Outer Membrane Protein F (OmpF) with specific solute selectivity.
- Ratul Chowdhury
- , Tingwei Ren
- & Costas D. Maranas
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Article
| Open AccessCarboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts
Previous efforts to assemble Rubisco within a cyanobacterial carboxysome-derived protein shell in plant chloroplasts to concentrate CO2 have been unsuccessful. Here, Long et al. produce carboxysomes in tobacco chloroplasts that encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2.
- Benedict M. Long
- , Wei Yih Hee
- & G. Dean Price
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Article
| Open AccessDe novo targeting to the cytoplasmic and luminal side of bacterial microcompartments
Bacterial microcompartments (BMCs) are protein-bound organelles encapsulating segments of metabolic pathways. Here the authors utilize specific de novo coiled-coil protein-protein interactions to display proteins on the outer or inner surface of BMCs.
- Matthew J. Lee
- , Judith Mantell
- & Martin J. Warren
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Article
| Open AccessConditional control of fluorescent protein degradation by an auxin-dependent nanobody
Current approaches to conditionally deplete target proteins require site-specific genetic engineering or have poor temporal control. Here the authors overcome these limitations by combining the AID system with nanobodies to reversibly degrade GFP-tagged proteins in living cells and zebrafish.
- Katrin Daniel
- , Jaroslav Icha
- & Jörg Mansfeld
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Article
| Open AccessSelective N-terminal acylation of peptides and proteins with a Gly-His tag sequence
His-tagged proteins can undergo N-terminal acylation as an undesired side-reaction. Here, the authors utilize this to develop a method for highly selective acylation and further modification of peptides and proteins using an optimized His sequence and 4-methoxyphenyl esters as acyl donors.
- Manuel C. Martos-Maldonado
- , Christian T. Hjuler
- & Knud J. Jensen
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Article
| Open AccessProgrammed loading and rapid purification of engineered bacterial microcompartment shells
Bacterial microcompartments are protein-bound organelles encapsulating segments of metabolic pathways. Here the authors functionalise shell proteins to facilitate facile purification and enable cargo encapsulation via covalent linkage.
- Andrew Hagen
- , Markus Sutter
- & Cheryl A. Kerfeld
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Article
| Open AccessHighly active enzymes by automated combinatorial backbone assembly and sequence design
Computationally designed enzymes often show lower activity or stability than their natural counterparts. Here, the authors present an evolution-inspired method for automated enzyme design, creating stable enzymes with accurate active site architectures and wild-type-like activities.
- Gideon Lapidoth
- , Olga Khersonsky
- & Sarel J. Fleishman
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Article
| Open AccessModular ssDNA binding and inhibition of telomerase activity by designer PPR proteins
Pentatricopeptide repeat proteins bind single-stranded RNA and have been used to study ssRNA biology. Here the authors co-opt these proteins to target ssDNA and demonstrate specific binding of telomere sequences, the structural basis for ssDNA wrapping, and use them as potent telomerase inhibitors.
- Henrik Spåhr
- , Tiongsun Chia
- & Oliver Rackham
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| Open AccessIn silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design
Antimicrobial peptides are considered promising alternatives to antibiotics. Here the authors developed a computational algorithm that starts with peptides naturally occurring in plants and optimizes this starting material to yield new variants which are highly distinct from the parent peptide.
- William F. Porto
- , Luz Irazazabal
- & Octavio L. Franco
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Article
| Open AccessDirected evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
CXCR2 antagonism has been shown to be anti-arthritic, but anti-chemokine therapies usually fail in the clinic owing to redundancy in chemokine-receptor interactions. Here the authors develop single-chain antibodies with multiple chemokine specificities to achieve high affinity and broad specificity to mouse and human CXC chemokines with efficacy in a K/BxN serum transfer model of arthritis.
- Alessandro Angelini
- , Yoshishige Miyabe
- & K. Dane Wittrup
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Article
| Open AccessEngineering enhanced cellobiohydrolase activity
Cellobiohydrolases (CBHs) are critical for natural and industrial biomass degradation but their structure–activity relationships are not fully understood. Here, the authors present the biochemical and structural characterization of two CBHs, identifying protein regions that confer enhanced CBH activity.
- Larry E. Taylor II
- , Brandon C. Knott
- & Gregg T. Beckham
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Article
| Open AccessEfficient molecular evolution to generate enantioselective enzymes using a dual-channel microfluidic droplet screening platform
Optimizing an enzyme usually requires testing thousands of variants, thus consuming large amounts of material and time. Here, the authors present a method that allows for measuring two different activities of the same enzyme simultaneously instead of doing two consecutive rounds of screening.
- Fuqiang Ma
- , Meng Ting Chung
- & Guang-Yu Yang
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Article
| Open AccessTwo distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase
The substrate specificity of acyl-ACP thioesterase (TE) plays a crucial role in determining the product profile of type II fatty acid synthase. Utilizing two FatB-type acyl-ACP TEs, the authors here define determinants of substrate specificity and create synthetic enzymes with distinct catalytic traits.
- Fuyuan Jing
- , Le Zhao
- & Basil J. Nikolau
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Article
| Open AccessAntimicrobial peptide capsids of de novo design
With the growing threat of antibiotic resistance, unconventional approaches to antimicrobial discovery are needed. Here, the authors present a peptide topology that mimics virus architecture and assembles into antimicrobial capsids that disrupt bacterial membranes upon contact.
- Emiliana De Santis
- , Hasan Alkassem
- & Maxim G. Ryadnov
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Article
| Open AccessMammalian display screening of diverse cystine-dense peptides for difficult to drug targets
Pathologies related to protein:protein interaction are hard to treat but cystine-dense peptides have the potential to disrupt such interactions. Here the authors develop a high-diversity mammalian cell screen for cystine-dense peptides with drug potential and use it to identify a YAP:TEAD inhibitor.
- Zachary R. Crook
- , Gregory P. Sevilla
- & James M. Olson
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Article
| Open AccessEngineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system
G-protein-coupled receptors are a large and diverse group of eukaryotic membrane receptors. Here the authors couple GPCRs to dCas9 to link extracellular sensing to genome regulation.
- Nathan H. Kipniss
- , P. C. Dave P. Dingal
- & Lei S. Qi
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Article
| Open AccessIdentifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer
E3 ubiquitin ligases regulate biological functions by ubiquitinating defined substrate proteins but overlapping specificities complicate the identification of E3-substrate relationships. Here, the authors construct an orthogonal UB transfer cascade and identify specific substrates of the E3 enzyme E6AP.
- Yiyang Wang
- , Xianpeng Liu
- & Jun Yin
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Article
| Open AccessEvolutionary action and structural basis of the allosteric switch controlling β2AR functional selectivity
Ligand-induced biased signaling is thought to result in part from ligand-specific receptor conformations that cause the engagement of distinct effectors. Here the authors trace and evaluate the impact of mutations of the β2–adrenergic receptor on multiple signaling outputs to provide structural-level insight into the determinants of GPCR functional selectivity.
- Anne-Marie Schönegge
- , Jonathan Gallion
- & Michel Bouvier
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Article
| Open AccessRestoration of patterned vision with an engineered photoactivatable G protein-coupled receptor
To restore sight after retinal degeneration, one approach is to express light-sensitive proteins in remaining cells. Here the authors combine a light-sensitive engineered G protein-coupled receptor and ion channels to restore ON and OFF responses as well as superior visual pattern discrimination.
- Michael H. Berry
- , Amy Holt
- & Ehud Y. Isacoff
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Article
| Open AccessPhage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein
Lanthipeptides are a class of cyclic post-translationally modified peptides with potential drug-like properties. Here the authors develop a phage display system by expressing lanthipeptide precursors as C-terminal fusions to the phage M13 coat protein pIII in E. coli along with the heterologous modifying enzymes.
- Johannes H. Urban
- , Markus A. Moosmeier
- & Josef Prassler
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Article
| Open AccessEngineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors
A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients.
- Daniël Verhoef
- , Koen M. Visscher
- & Mettine H. A. Bos
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Article
| Open AccessTowards designer organelles by subverting the peroxisomal import pathway
Designer organelles could allow the isolation of synthetic biological pathways from endogenous components of the host cell. Here the authors engineer a peroxisomal protein import pathway orthogonal to the naturally occurring system.
- Laura L. Cross
- , Rupesh Paudyal
- & Stuart L. Warriner
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Article
| Open AccessImproved split fluorescent proteins for endogenous protein labeling
Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.
- Siyu Feng
- , Sayaka Sekine
- & Bo Huang
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Article
| Open AccessConstruction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme
Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2.
- Daniel W. Watkins
- , Jonathan M. X. Jenkins
- & J. L. Ross Anderson