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Structure- and sequence-based design of synthetic single-domain antibody libraries Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-12-21 Alexander M Sevy; Ming-Tang Chen; Michelle Castor; Tyler Sylvia; Harini Krishnamurthy; Andrii Ishchenko; Chung-Ming Hsieh
Single-domain antibody fragments known as VHH have emerged in the pharmaceutical industry as useful biotherapeutics. These molecules, which are naturally produced by camelids, share the characteristics of high affinity and specificity with traditional human immunoglobulins, while consisting of only a single heavy chain. Currently, the most common method for generating VHH is via animal immunization
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Protein Engineering, Design and Selection Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-10-23 Roberto A Chica
The 2020 volume of Protein Engineering, Design and Selection (PEDS) marks the beginning of my appointment as Editor-in-Chief of the journal. Since its foundation in 1986, PEDS has built a strong reputation as a respected publishing destination for our research community. I am thrilled to be given the opportunity to build on the journal’s legacy of rigorous and constructive manuscript review by leading
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A novel phage display vector for selection of target-specific peptides Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-10-03 Alex Chang; Joey P Ting; Alfonso Espada; Howard Broughton; Manuel Molina-Martin; Sepideh Afshar
Intrinsic low display level of polypeptides on phage is a fundamental and limiting hurdle in successful isolation of target-specific binders by phage display technology. To circumvent this challenge, we optimized the copy number of peptides displayed on the phage surface using type 33 phage vector. We randomized the first 67 amino acids of the wild type PIII to identify mutants that would result in
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Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-09-14 Majid Masso
A computational mutagenesis technique was used to characterize the structural effects associated with over 46 000 single and multiple amino acid variants of Aequorea victoria green fluorescent protein (GFP), whose functional effects (fluorescence levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall change in sequence-structure
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Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-09-14 P L Herzog,E Borghi,M W Traxlmayr,C Obinger,H D Sikes,C K Peterbauer
Accurate yet efficient high-throughput screenings have emerged as essential technology for enzyme engineering via directed evolution. Modern high-throughput screening platforms for oxidoreductases are commonly assisted by technologies such as surface display and rely on emulsification techniques to facilitate single-cell analysis via fluorescence-activated cell sorting. Empowered by the dramatically
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Engineering a fluorescence biosensor for the herbicide glyphosate. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-09-14 Pierre-Emmanuel Y N'Guetta,Maggie M Fink,Shahir S Rizk
Glyphosate, the active ingredient in RoundUp, is the most widely used herbicide on the globe, and has recently been linked to an increased risk in non-Hodgkin’s lymphoma in exposed individuals. Therefore, detection and monitoring of glyphosate levels in water and soil is important for public safety. Here, we describe a biosensor for glyphosate based on an engineered Escherichia coli phosphonate-binding
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Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-09-14 Subba Reddy Dodda,Nibedita Sarkar,Piyush Jain,Kaustav Aikat,Sudit S Mukhopadhyay
Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated
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Understanding microcystin-LR antibody binding interactions using in silico docking and in vitro mutagenesis. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-07-29 Daniel A McPartlin,Caroline Murphy,Jenny Fitzgerald,Hui Ma,Fiona Regan,Richard J O'Kennedy
Microcystins (MCs) are a group of highly potent cyanotoxins that are becoming more widely distributed due to increased global temperatures and climate change. Microcystin-leucine-arginine (MC-LR) is the most potent and most common variant, with a guideline limit of 1 μg/l in drinking water. We previously developed a novel avian single-chain fragment variable (scFv), designated 2G1, for use in an optical-planar
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Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-07-29 Magdalena Wójcik,Susana Vázquez Torres,Wim J Quax,Ykelien L Boersma
Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many
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Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-07-29 Sara M O'Rourke,Giora I Morozov,Jacob T Roberts,Adam W Barb,Nikolaos G Sgourakis
Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I
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Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-07-29 Marcia Stickler,Anita Reddy,Joanna M Xiong,Melanie H Wong,Yoshiko Akamatsu,Paul R Hinton,Fiona A Harding
Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created
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Conformational selection of allergen-antibody complexes-surface plasticity of paratopes and epitopes. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-07-28 Monica L Fernández-Quintero,Johannes R Loeffler,Franz Waibl,Anna S Kamenik,Florian Hofer,Klaus R Liedl
Antibodies have the ability to bind various types of antigens and to recognize different antibody-binding sites (epitopes) of the same antigen with different binding affinities. Due to the conserved structural framework of antibodies, their specificity to antigens is mainly determined by their antigen-binding site (paratope). Therefore, characterization of epitopes in combination with describing the
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CodonAdjust: a software for in silico design of a mutagenesis library with specific amino acid profiles. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-07-24 Thuy Duong Nguyen,Yutaka Saito,Tomoshi Kameda
In protein engineering, generation of mutagenesis libraries is a key step to study the functions of mutants. To generate mutants with a desired composition of amino acids (AAs), a codon consisting of a mixture of nucleotides is widely applied. Several computational methods have been proposed to calculate a codon nucleotide composition for generating a given amino acid profile based on mathematical
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Protein tolerance to random circular permutation correlates with thermostability and local energetics of residue-residue contacts. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-07-06 Joshua T Atkinson,Alicia M Jones,Vikas Nanda,Jonathan J Silberg
Adenylate kinase (AK) orthologs with a range of thermostabilities were subjected to random circular permutation, and deep mutational scanning was used to evaluate where new protein termini were nondisruptive to activity. The fraction of circularly permuted variants that retained function in each library correlated with AK thermostability. In addition, analysis of the positional tolerance to new termini
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In vitro evolution of phi29 DNA polymerases through compartmentalized gene expression and rolling-circle replication. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-06-13 Yoshihiro Sakatani,Ryo Mizuuchi,Norikazu Ichihashi
Phi29 DNA polymerase is widely used for DNA amplification through rolling-circle replication or multiple displacement amplification. Here, we performed completely in vitro artificial evolution of phi29 DNA polymerase by combining the in vitro compartmentalization and the gene expression-coupled rolling-circle replication of a circular DNA encoding the polymerase. We conducted the experiments in six
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Dissecting the statistical properties of the linear extrapolation method of determining protein stability. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Kresten Lindorff-Larsen
The linear extrapolation method to determine protein stability from denaturant-induced unfolding experiments is based on the observation that the free energy of unfolding is often a linear function of the denaturant concentration. The value in the absence of denaturant is then estimated by extrapolation from this linear relationship. Parameters and their confidence intervals are typically estimated
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Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Ichiro Matsumura,Donian Chyong
The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, 'ribosome pegging', to exclude common unwanted mutations that benefit host cells at the expense of heterologous
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Computer-guided library generation applied to the optimization of single-domain antibodies. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-03-13 Hiroki Akiba,Hiroko Tamura,Jose M M Caaveiro,Kouhei Tsumoto
Computer-guided library generation is a plausible strategy to optimize antibodies. Herein, we report the improvement of the affinity of a single-domain camelid antibody for its antigen using such approach. We first conducted experimental and computational alanine scanning to describe the precise energetic profile of the antibody–antigen interaction surface. Based on this characterization, we hypothesized
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Antibody humanization-the Influence of the antibody framework on the CDR-H3 loop ensemble in solution. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-03-04 Monica L Fernández-Quintero,Martin C Heiss,Klaus R Liedl
Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the
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Molecular dynamics study of ACBP denaturation in alkyl sulfates demonstrates possible pathways of unfolding through fused surfactant clusters. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-02 Armen H Poghosyan,Nicholas P Schafer,Jeppe Lyngsø,Aram A Shahinyan,Jan Skov Pedersen,Daniel E Otzen
Anionic surfactants denature proteins at low millimolar concentrations, yet little is known about the underlying molecular mechanisms. Here, we undertake 1-μs-long atomistic molecular dynamics simulations of the denaturation of acyl coenzyme A binding protein (ACBP) and compare our results with previously published and new experimental data. Since increasing surfactant chain length is known to lead
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A mixture of three engineered phosphotriesterases enables rapid detoxification of the entire spectrum of known threat nerve agents. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-10-15 Dragana Despotović,Einav Aharon,Artem Dubovetskyi,Haim Leader,Yacov Ashani,Dan S Tawfik
Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase
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Erratum to: Affinity versus specificity in coupled binding and folding reactions. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-10-06 Stefano Gianni,Per Jemth
In the above article the symbols for ‘nanomolar’ and ‘micromolar’ were incorrectly expanded during typesetting to read ‘nanometer’ and ‘micrometer’ respectively. These have now been corrected to reflect their original intended meaning.
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Information theoretic measures for quantifying sequence-ensemble relationships of intrinsically disordered proteins. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-08-03 Megan C Cohan,Kiersten M Ruff,Rohit V Pappu
Intrinsically disordered proteins (IDPs) contribute to a multitude of functions. De novo design of IDPs should open the door to modulating functions and phenotypes controlled by these systems. Recent design efforts have focused on compositional biases and specific sequence patterns as the design features. Analysis of the impact of these designs on sequence-function relationships indicates that individual
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Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-03-02 Magdalena J Buschhaus,Stefan Becker,Andrew J Porter,Caroline J Barelle
The adaptive immune system of cartilaginous fish (Elasmobranchii), comprising of classical hetero-tetrameric antibodies, is enhanced through the presence of a naturally occurring homodimeric antibody-like immunoglobulin-the new antigen receptor (IgNAR). The binding site of the IgNAR variable single-domain (VNAR) offers advantages of reduced size (<1/10th of classical immunoglobulin) and extended binding
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Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-02-25 Stefan Jarl Christensen,Silke Flindt Badino,Ana Mafalda Cavaleiro,Kim Borch,Peter Westh
The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity
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Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/β protein. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-02-22 Matthew Gill,Michelle E McCully
Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase
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Engineered variants of a lipase from Yarrowia lipolytica with improved trypsin resistance for enzyme replacement therapy. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Huitu Zhang,Huan Liu,Ying Zhang,Tongwei Sun,Guoguo Wu,Cuixia Zhou,Xiaonong Wu,Jing Zhang,Rong Yue,Haikuan Wang,Yujie Dai,Fufeng Liu,Fuping Lu
To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica, the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved
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Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Tanja Gerlza,Michael Nagele,Martha Gschwandtner,Sophie Winkler,Andreas Kungl
The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered
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FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2020-01-11 Evelyn Eggenstein,Antonia Richter,Arne Skerra
FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B)
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Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Alexander Schmidt,Alexey Shvetsov,Elena Soboleva,Yury Kil,Vladimir Sergeev,Marina Surzhik
Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants
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Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-23 James T Heads,Richard Lamb,Sebastian Kelm,Ralph Adams,Peter Elliott,Kerry Tyson,Sarfaraj Topia,Shauna West,Ruodan Nan,Alison Turner,Alastair D G Lawson
Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having
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Attempts to develop an enzyme converting DHIV to KIV. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-23 Kenji Oki,Frederick S Lee,Stephen L Mayo
Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered
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Improved ligand-binding- and signaling-competent human NK2R yields in yeast using a chimera with the rat NK2R C-terminus enable NK2R-G protein signaling platform. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Abhinav R Jain,Zachary T Britton,Chester E Markwalter,Anne S Robinson
The tachykinin 2 receptor (NK2R) plays critical roles in gastrointestinal, respiratory and mental disorders and is a well-recognized target for therapeutic intervention. To date, therapeutics targeting NK2R have failed to meet regulatory agency approval due in large part to the limited characterization of the receptor-ligand interaction and downstream signaling. Herein, we report a protein engineering
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Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Filip Claes,Stanislav Rudyak,Angela S Laird,Nikolaos Louros,Jacinte Beerten,Maja Debulpaep,Emiel Michiels,Rob van der Kant,Joost Van Durme,Greet De Baets,Bert Houben,Meine Ramakers,Kristy Yuan,Serene S L Gwee,Sara Hernandez,Kerensa Broersen,Mikael Oliveberg,Barbara Moahamed,Janine Kirstein,Wim Robberecht,Frederic Rousseau,Joost Schymkowitz
The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral
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Another week, another breakthrough: immunotherapy. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 James S Huston
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Shared unfolding pathways of unrelated immunoglobulin-like β-sandwich proteins. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-23 Rudesh D Toofanny,Sara Calhoun,Amanda L Jonsson,Valerie Daggett
The Dynameomics project contains native state and unfolding simulations of 807 protein domains, where each domain is representative of a different metafold; these metafolds encompass ~97% of protein fold space. There is a long-standing question in structural biology as to whether proteins in the same fold family share the same folding/unfolding characteristics. Using molecular dynamics simulations
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RNA-seq-based identification of Star upregulation by islet amyloid formation. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-13 Meghan F Hogan,Mark Ziemann,Harikrishnan K N,Hanah Rodriguez,Antony Kaspi,Nathalie Esser,Andrew T Templin,Assam El-Osta,Steven E Kahn
Aggregation of islet amyloid polypeptide (IAPP) into islet amyloid results in β-cell toxicity in human type 2 diabetes. To determine the effect of islet amyloid formation on gene expression, we performed ribonucleic acid (RNA) sequencing (RNA-seq) analysis using cultured islets from either wild-type mice (mIAPP), which are not amyloid prone, or mice that express human IAPP (hIAPP), which develop amyloid
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Amyloidogenicity and cytotoxicity of des-Lys-1 human amylin provides insight into amylin self-assembly and highlights the difficulties of defining amyloidogenicity. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-13 Kyung-Hoon Lee,Alexander Zhyvoloup,Daniel Raleigh
The polypeptide amylin is responsible for islet amyloid in type 2 diabetes, a process which contributes to β-cell death in the disease. The role of the N-terminal region of amylin in amyloid formation is relatively unexplored, although removal of the disulfide bridged loop between Cys-2 and Cys-7 accelerates amyloid formation. We examine the des Lys-1 variant of human amylin (h-amylin), a variant which
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Loss of perlecan heparan sulfate glycosaminoglycans lowers body weight and decreases islet amyloid deposition in human islet amyloid polypeptide transgenic mice. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-13 Andrew T Templin,Mahnaz Mellati,Raija Soininen,Meghan F Hogan,Nathalie Esser,J Josh Castillo,Sakeneh Zraika,Steven E Kahn,Rebecca L Hull
Islet amyloid is a pathologic feature of type 2 diabetes (T2D) that is associated with β-cell loss and dysfunction. These amyloid deposits form via aggregation of the β-cell secretory product islet amyloid polypeptide (IAPP) and contain other molecules including the heparan sulfate proteoglycan perlecan. Perlecan has been shown to bind amyloidogenic human IAPP (hIAPP) via its heparan sulfate glycosaminoglycan
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The N-terminal 1-55 residues domain of pyruvate dehydrogenase from Escherichia coli assembles as a dimer in solution. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Yuanyuan Wang,Zemao Gong,Han Fang,Dongming Zhi,Hu Tao
The pyruvate dehydrogenase complex (PDHc) from Escherichia coli is a large protein complex consisting of multiple copies of the pyruvate dehydrogenase (E1ec), dihydrolipoamide acetyltransferase (E2ec) and dihydrolipoamide dehydrogenase (E3ec). The N-terminal domain (NTD, residues 1-55) of E1ec plays a critical role in the interaction between E1ec and E2ec and the whole PDHc activity. Using circular
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A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-31 Jason M Lajoie,Yong Ku Cho,Dustin Frost,Samantha Bremner,Lingjun Li,Eric V Shusta
Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target
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Development of a novel prostate apoptosis response-4 (Par-4) protein entity with an extended duration of action for therapeutic treatment of cancer. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-13 Kyungbo Kim,Pereira Araujo,Nikhil Hebbar,Ziyuan Zhou,Xirong Zheng,Fang Zheng,Vivek M Rangnekar,Chang-Guo Zhan
Prostate apoptosis response-4 (Par-4) is a tumor suppressor which protects against neoplastic transformation. Remarkably, Par-4 is capable of inducing apoptosis selectively in cancer cells without affecting the normal cells. In this study, we found that recombinant Par-4 protein had limited serum persistence in mice that may diminish its anti-tumor activity in vivo. To improve the in vivo performance
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Antibody discovery and the arrow of time. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2019-12-13 James S Huston
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Engineering of the upper hinge region of human IgG1 Fc enhances the binding affinity to FcγIIIa (CD16a) receptor isoform. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-10-10 Dana N Ashoor,Noureddine Ben Khalaf,Sonia Bourguiba-Hachemi,Maryam H Marzouq,M Dahmani Fathallah
The interaction between antibodies and Immune cells surface FcγRIIIa (CD16a) receptor triggers a variety of immune responses including antibody-dependent cell-mediated cytotoxicity, antibody neutralization, phagocytosis, inflammation and tissue injury. Recent studies showed that IgG1 upper hinge region and FcγRs polymorphism play a major role in the interaction with Fcγ receptors and in the stability
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Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-09-29 Michael J Rudolph,David J Vance,Simon Kelow,Siva Krishna Angalakurthi,Sophie Nguyen,Simon A Davis,Yinghui Rong,C Russell Middaugh,David D Weis,Roland Dunbrack,John Karanicolas,Nicholas J Mantis
Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed
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Adnectin-drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-09-25 Daša Lipovšek,Irvith Carvajal,Alban J Allentoff,Anthony Barros,John Brailsford,Qiang Cong,Pete Cotter,Sanjeev Gangwar,Cris Hollander,Virginie Lafont,Wai Leung Lau,Wenying Li,Miguel Moreta,Steven O'Neil,Jason Pinckney,Michael J Smith,Julie Su,Christina Terragni,Michael A Wallace,Lifei Wang,Martin Wright,H Nicholas Marsh,James W Bryson
Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody-drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens-but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin-drug conjugates, an alternative
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Site-directed mutagenesis: role of lid region for T1 lipase specificity. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-09-22 Rauda A Mohamed,Abu Bakar Salleh,Thean Chor Leow,Normi M Yahaya,Mohd Basyaruddin Abdul Rahman
A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable
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New engineered phenolic biosensors based on the AraC regulatory protein. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-09-22 C S Frei,S Qian,P C Cirino
Customized transcription factors that control gene expression in response to small molecules can act as endogenous molecular biosensors and are valuable tools for synthetic biology. We previously engineered the Escherichia coli regulatory protein AraC to respond to non-native inducers such as D-arabinose and triacetic acid lactone. Those prior studies involved the construction and screening of individual
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Rational optimization of a monoclonal antibody for simultaneous improvements in its solution properties and biological activity. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-09-07 Sandeep Kumar,Kirk Roffi,Dheeraj S Tomar,David Cirelli,Nicholas Luksha,Danielle Meyer,Jeffrey Mitchell,Martin J Allen,Li Li
Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of
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Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-09-01 S-Y Lau,J W Siau,R M Sobota,C-I Wang,P Zhong,D P Lane,F J Ghadessy
Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay
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Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-09-01 Joerg Thomas Regula,Sabine Imhof-Jung,Michael Mølhøj,Joerg Benz,Andreas Ehler,Alexander Bujotzek,Wolfgang Schaefer,Christian Klein
Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach
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Analysis of nanobody paratopes reveals greater diversity than classical antibodies. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-07-28 Laura S Mitchell,Lucy J Colwell
Nanobodies (Nbs) are a class of antigen-binding protein derived from camelid immune systems, which achieve equivalent binding affinities and specificities to classical antibodies (Abs) despite being comprised of only a single variable domain. Here, we use a data set of 156 unique Nb:antigen complex structures to characterize Nb-antigen binding and draw comparison to a set of 156 unique Ab:antigen structures
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De novo designed transmembrane peptides activating the α5β1 integrin. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-07-12 Marco Mravic,Hailin Hu,Zhenwei Lu,Joel S Bennett,Charles R Sanders,A Wayne Orr,William F DeGrado
Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbβ3 and αvβ3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5β1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our
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Molecular dynamics-derived rotamer libraries for d-amino acids within homochiral and heterochiral polypeptides. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-07-12 Matthew Carter Childers,Clare-Louise Towse,Valerie Daggett
Computational resources have contributed to the design and engineering of novel proteins by integrating genomic, structural and dynamic aspects of proteins. Non-canonical amino acids, such as d-amino acids, expand the available sequence space for designing and engineering proteins; however, the rotamer libraries for d-amino acids are usually constructed as the mirror images of l-amino acid rotamer
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A novel dual-cytokine-antibody fusion protein for the treatment of CD38-positive malignancies. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-07-10 Roberto De Luca,Paul Kachel,Klara Kropivsek,Berend Snijder,Markus G Manz,Dario Neri
A novel dual-cytokine-antibody fusion protein, consisting of an antibody directed against CD38 [a tumor-associated antigen mainly expressed on the surface of multiple myeloma (MM) cells], simultaneously fused to both tumor necrosis factor ligand superfamily member 10 (TRAIL) and interleukin-2 (IL2), was designed, expressed and purified to homogeneity. The novel fusion protein, termed IL2-αCD38-αCD38-scTRAIL
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Antibody discovery and the arrow of time. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-07-01 James S Huston
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The structure of SALM5 suggests a dimeric assembly for the presynaptic RPTP ligand recognition. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-06-14 Sudeep Karki,Prodeep Paudel,Celeste Sele,Alexander V Shkumatov,Tommi Kajander
Synaptic adhesion molecules play a crucial role in the regulation of synapse development and maintenance. Recently, several families of leucine-rich repeat (LRR) domain-containing neuronal adhesion molecules have been characterised, including netrin-G ligands, LRRTMs and the synaptic adhesion-like molecule (SALM) family proteins. Most of these are expressed at the excitatory glutamatergic synapses
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Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-06-14 Jennifer I Lai,Deeptak Verma,Chris Bailey-Kellogg,Margaret E Ackerman
Structure-based approaches to antigen design utilize insights from antibody (Ab):antigen interactions and a refined understanding of protective Ab responses to engineer novel antigens presenting epitopes with conformations relevant to eliciting or discovering protective humoral responses. For human immunodeficiency virus-1 (HIV-1), one model of protection is provided by broadly neutralizing Abs (bnAbs)
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A platform for chemical modification of mandelate racemase: characterization of the C92S/C264S and γ-thialysine 166 variants. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-06-01 Mitesh Nagar,Himank Kumar,Stephen L Bearne
Mandelate racemase (MR) serves as a paradigm for our understanding of enzyme-catalyzed deprotonation of a carbon acid substrate. To facilitate structure-function studies on MR using non-natural amino acid substitutions, we engineered the Cys92Ser/Cys264Ser variant (dmMR) as a platform for introducing Cys residues at specific locations for subsequent covalent modification. While the highly reactive
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Roles of the disulfide bond between the variable and the constant domains of rabbit immunoglobulin kappa chains in thermal stability and affinity. Protein Eng. Des. Sel. (IF 1.774) Pub Date : 2018-06-01 Raiji Kawade,Hiroki Akiba,Kevin Entzminger,Toshiaki Maruyama,C J Okumura,Kouhei Tsumoto
Rabbit antibodies show unique structural characteristics in that kappa chains have an inter-domain disulfide bond between the variable and constant domains. Here we characterized this disulfide bond from physicochemical viewpoints both in stability and affinity. It was revealed that the disulfide bond contributed to the thermal stability of the antibody, but the affinity and mechanism of antigen recognition
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