Protein Engineering Design & Selection最新文献_第10页

Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity. 通过增强GAG结合亲和力设计改进的t细胞动员CXCL10突变体。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz043

Tanja Gerlza, Michael Nagele, Martha Gschwandtner, Sophie Winkler, Andreas Kungl

{"title":"Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity.","authors":"Tanja Gerlza, Michael Nagele, Martha Gschwandtner, Sophie Winkler, Andreas Kungl","doi":"10.1093/protein/gzz043","DOIUrl":"https://doi.org/10.1093/protein/gzz043","url":null,"abstract":"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 human CXCL10 towards improved T-cell mobilisation by implementing a single site-directed mutation N20K into the protein, which leads to a higher GAG binding affinity compared to the wild type. Interestingly, this mutation not only increased T-cell migration in a transendothelial migration assay, the mutant intensified T-cell chemotaxis also in a Boyden chamber set-up thereby indicating a strong role of T-cell-localised GAGs on leukocyte migration. A CXCL10 mutant with increased GAG-binding affinity could therefore potentially serve as a T-cell mobiliser in pathological conditions where the immune surveillance of the target tissue is impaired, as is the case for most solid tumors.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 8","pages":"367-373"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz043","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37572249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability. 静电相互作用调节IgG1和IgG4P抗体的不同聚集倾向，并通知带电残基取代以提高可发育性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz046

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

{"title":"Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability.","authors":"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","doi":"10.1093/protein/gzz046","DOIUrl":"https://doi.org/10.1093/protein/gzz046","url":null,"abstract":"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 identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 6","pages":"277-288"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37484111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 20

Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design. 通过共识设计获得的具有改进的蛋白质热稳定性和酶活性的分选酶突变体。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa018

Magdalena Wójcik, Susana Vázquez Torres, Wim J Quax, Ykelien L Boersma

引用次数: 9

FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection. FluoroCalins:具有新型结合功能与荧光蛋白融合的工程脂钙蛋白，用于生物分子成像和检测。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz047

Evelyn Eggenstein, Antonia Richter, Arne Skerra

{"title":"FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection.","authors":"Evelyn Eggenstein, Antonia Richter, Arne Skerra","doi":"10.1093/protein/gzz047","DOIUrl":"https://doi.org/10.1093/protein/gzz047","url":null,"abstract":"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) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 6","pages":"289-296"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37533945","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Engineered variants of a lipase from Yarrowia lipolytica with improved trypsin resistance for enzyme replacement therapy. 酶替代疗法中改良胰蛋白酶抵抗的脂肪酶工程变体。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa001

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

引用次数: 1

Understanding microcystin-LR antibody binding interactions using in silico docking and in vitro mutagenesis. 利用硅对接和体外诱变了解微囊藻毒素- lr抗体结合相互作用。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa016

Daniel A McPartlin, Caroline Murphy, Jenny Fitzgerald, Hui Ma, Fiona Regan, Richard J O'Kennedy

{"title":"Understanding microcystin-LR antibody binding interactions using in silico docking and in vitro mutagenesis.","authors":"Daniel A McPartlin, Caroline Murphy, Jenny Fitzgerald, Hui Ma, Fiona Regan, Richard J O'Kennedy","doi":"10.1093/protein/gzaa016","DOIUrl":"https://doi.org/10.1093/protein/gzaa016","url":null,"abstract":"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 waveguide detection system for microcystin determination. This current work investigates interactions between 2G1 and MC-LR at the molecular level through modelling with an avian antibody template and molecular docking by AutoDock Vina to identify key amino acid (AA) residues involved. These potential AA interactions were investigated in vitro by targeted mutagenesis, specifically, by alanine scanning mutations. Glutamic acid (E) was found to play a critical role in the 2G1-MC-LR binding interaction, with the heavy chain glutamic acid (E) 102 (H-E102) forming direct bonds with the arginine (R) residue of MC-LR. In addition, alanine mutation of light chain residue aspartic acid 57 (L-D57) led to an improvement in antigen-binding observed using enzyme-linked immunosorbent assay (ELISA), and was confirmed by surface plasmon resonance (SPR). This work will contribute to improving the binding of recombinant anti-MC-LR to its antigen and aid in the development of a higher sensitivity harmful algal toxin diagnostic.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 12","pages":"533-542"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa016","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38203299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

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. 利用与大鼠NK2R c端嵌合体，在酵母中提高配体结合和信号能力的人NK2R产量，使NK2R- g蛋白信号传导平台成为可能。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa009

Abhinav R Jain, Zachary T Britton, Chester E Markwalter, Anne S Robinson

{"title":"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.","authors":"Abhinav R Jain, Zachary T Britton, Chester E Markwalter, Anne S Robinson","doi":"10.1093/protein/gzaa009","DOIUrl":"https://doi.org/10.1093/protein/gzaa009","url":null,"abstract":"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 strategy to improve ligand-binding- and signaling-competent human NK2R that enables a yeast-based NK2R signaling platform by creating chimeras utilizing sequences from rat NK2R. We demonstrate that NK2R chimeras incorporating the rat NK2R C-terminus exhibited improved ligand-binding yields and downstream signaling in engineered yeast strains and mammalian cells, where observed yields were better than 4-fold over wild type. This work builds on our previous studies that suggest exchanging the C-termini of related and well-expressed family members may be a general protein engineering strategy to overcome limitations to ligand-binding and signaling-competent G protein-coupled receptor yields in yeast. We expect these efforts to result in NK2R drug candidates with better characterized signaling properties.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 10","pages":"459-469"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa009","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37930375","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Affinity versus specificity in coupled binding and folding reactions. 偶联结合和折叠反应的亲和与特异性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz020

S. Gianni, P. Jemth

{"title":"Affinity versus specificity in coupled binding and folding reactions.","authors":"S. Gianni, P. Jemth","doi":"10.1093/protein/gzz020","DOIUrl":"https://doi.org/10.1093/protein/gzz020","url":null,"abstract":"Intrinsically disordered protein regions may fold upon binding to an interaction partner. It is often argued that such coupled binding and folding enables the combination of high specificity with low affinity. The basic tenet is that an unfavorable folding equilibrium will make the overall binding weaker while maintaining the interaction interface. While theoretically solid, we argue that this concept may be misleading for intrinsically disordered proteins. In fact, experimental evidence suggests that interactions of disordered regions usually involve extended conformations. In such cases, the disordered region is exceptionally unlikely to fold into a bound conformation in the absence of its binding partner. Instead, these disordered regions can bind to their partners in multiple different conformations and then fold into the native bound complex, thus, if anything, increasing the affinity through folding. We concede that (de)stabilization of native structural elements such as helices will modulate affinity, but this could work both ways, decreasing or increasing the stability of the complex. Moreover, experimental data show that intrinsically disordered binding regions display a range of affinities and specificities dictated by the particular side chains and length of the disordered region and not necessarily by the fact that they are disordered. We find it more likely that intrinsically disordered regions are common in protein-protein interactions because they increase the repertoire of binding partners, providing an accessible route to evolve interactions rather than providing a stability-affinity trade-off.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"38 1","pages":""},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78364783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

P329G-CAR-J: a novel Jurkat-NFAT-based CAR-T reporter system recognizing the P329G Fc mutation. P329G- car - j:一种新的基于jurkat - nfat的识别P329G Fc突变的CAR-T报告系统。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz027

D. Darowski, C. Jost, K. Stubenrauch, Uwe Wessels, J. Benz, A. Ehler, A. Freimoser-Grundschober, P. Brünker, E. Mössner, P. Umaña, S. Kobold, C. Klein

{"title":"P329G-CAR-J: a novel Jurkat-NFAT-based CAR-T reporter system recognizing the P329G Fc mutation.","authors":"D. Darowski, C. Jost, K. Stubenrauch, Uwe Wessels, J. Benz, A. Ehler, A. Freimoser-Grundschober, P. Brünker, E. Mössner, P. Umaña, S. Kobold, C. Klein","doi":"10.1093/protein/gzz027","DOIUrl":"https://doi.org/10.1093/protein/gzz027","url":null,"abstract":"Monoclonal antibody-based therapeutics are an integral part of treatment of different human diseases, and the selection of suitable antibody candidates during the discovery phase is essential. Here, we describe a novel, cellular screening approach for the identification and characterization of therapeutic antibodies suitable for conversion into T cell bispecific antibodies using chimeric antigen receptor (CAR) transduced Jurkat-NFAT-luciferase reporter cells (CAR-J). For that purpose, we equipped a Jurkat-NFAT reporter cell line with a universal CAR, based on a monoclonal antibody recognizing the P329G mutation in the Fc-part of effector-silenced human IgG1-antibodies. In addition to scFv-based second generation CARs, Fab-based CARs employing the P329G-binder were generated. Using these anti-P329G-CAR-J cells together with the respective P329G-mutated IgG1-antibodies, we established a system, which facilitates the rapid testing of therapeutic antibody candidates in a flexible, high throughput setting during early stage discovery. We show that both, scFv- and Fab-based anti-P329G-CAR-J cells elicit a robust and dose-dependent luciferase signal if the respective antibody acts as an adaptor between tumor target and P329G-CAR-J cells. Importantly, we could demonstrate that functional characteristics of the antibody candidates, derived from the anti-P329G-CAR-J screening assay, are predictive for the functionality of these antibodies in the T cell bispecific antibody format.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"45 1","pages":""},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77433687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils. 未折叠的Cu/Zn超氧化物歧化酶在疏水表面的吸附催化其淀粉样原纤维的形成。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-13 DOI: 10.1093/protein/gzz033

Mohammad Ashhar I Khan, Ulrich Weininger, Sven Kjellström, Shashank Deep, Mikael Akke

{"title":"Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils.","authors":"Mohammad Ashhar I Khan, Ulrich Weininger, Sven Kjellström, Shashank Deep, Mikael Akke","doi":"10.1093/protein/gzz033","DOIUrl":"https://doi.org/10.1093/protein/gzz033","url":null,"abstract":"Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 2","pages":"77-85"},"PeriodicalIF":2.4,"publicationDate":"2019-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz033","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37453689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0