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

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

Kinetic trapping in protein folding. 蛋白质折叠中的动力学捕获。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-13 DOI: 10.1093/protein/gzz018

Angela E. Varela, Kevin A. England, S. Cavagnero

引用次数: 8

Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli. 高生物活性重组人骨形态发生蛋白-2在大肠杆菌中的可溶性表达及密码子优化纯化。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-13 DOI: 10.1093/protein/gzz028

Wei Chen, Caiqian Zhang, Yeqing Wu, Xiuping Su

{"title":"Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli.","authors":"Wei Chen, Caiqian Zhang, Yeqing Wu, Xiuping Su","doi":"10.1093/protein/gzz028","DOIUrl":"https://doi.org/10.1093/protein/gzz028","url":null,"abstract":"We developed a simple method of preparing recombinant human bone morphogenetic protein-2 (rhBMP-2) with high biological activity. This rhBMP-2 was overproduced in Escherichia coli as a fusion protein with thioredoxin 6xHis-tag at its amino terminus. The cDNA fragment of human bone morphogenetic protein-2 (hBMP-2) fused to the secretion signal of alkaline phosphatase (PhoA) was expressed under T7 promoter in E. coli. After DNA sequence confirmation, the recombinant vector pETpho-bmp2 was transformed into E. coli BL21 (DE3). rhBMP-2 was produced by the recombinant strain pETpho-bmp2/BL21 (DE3) in a soluble form with an yield of 6.2 mg/L culture. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) results showed that the molecular weight of the product was approximately 28 kD. Moreover, rhBMP-2 was secreted as a dimer with a natural structure. rhBMP-2, purified by Ni Nitrilotriacetic acid Agarose (Ni-NTA) affinity chromatography, was used to examine osteosarcoma MG-63 cells and assay the alkaline phosphatase (ALP) activity. Results showed that rhBMP-2 induced MG-63 cell differentiation. When the final concentration was 500 ng/mL, the effect was more remarkable and ALP activity reached 525% compared with that of the control group.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"19 1","pages":""},"PeriodicalIF":2.4,"publicationDate":"2019-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89880197","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

Improving folding properties of computationally designed proteins. 提高计算设计蛋白质的折叠性能。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-13 DOI: 10.1093/protein/gzz025

Benjamin Bjerre, J. Nissen, Mikkel Madsen, Jūratė Fahrig-Kamarauskaitė, R. K. Norrild, Peter C Holm, M. K. Nordentoft, C. O'Shea, M. Willemoës, K. E. Johansson, J. Winther

{"title":"Improving folding properties of computationally designed proteins.","authors":"Benjamin Bjerre, J. Nissen, Mikkel Madsen, Jūratė Fahrig-Kamarauskaitė, R. K. Norrild, Peter C Holm, M. K. Nordentoft, C. O'Shea, M. Willemoës, K. E. Johansson, J. Winther","doi":"10.1093/protein/gzz025","DOIUrl":"https://doi.org/10.1093/protein/gzz025","url":null,"abstract":"While the field of computational protein design has witnessed amazing progression in recent years, folding properties still constitute a significant barrier towards designing new and larger proteins. In order to assess and improve folding properties of designed proteins, we have developed a genetics-based folding assay and selection system based on the essential enzyme, orotate phosphoribosyl transferase from Escherichia coli. This system allows for both screening of candidate designs with good folding properties and genetic selection of improved designs. Thus, we identified single amino acid substitutions in two failed designs that rescued poorly folding and unstable proteins. Furthermore, when these substitutions were transferred into a well-structured design featuring a complex folding profile, the resulting protein exhibited native-like cooperative folding with significantly improved stability. In protein design, a single amino acid can make the difference between folding and misfolding, and this approach provides a useful new platform to identify and improve candidate designs.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"55 1","pages":""},"PeriodicalIF":2.4,"publicationDate":"2019-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74068137","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: 4

Molecular dynamics study of ACBP denaturation in alkyl sulfates demonstrates possible pathways of unfolding through fused surfactant clusters. 硫酸烷基酯中ACBP变性的分子动力学研究揭示了通过熔融表面活性剂团簇展开的可能途径。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-02 DOI: 10.1093/protein/gzz037

A. Poghosyan, Nicholas P. Schafer, Jeppe Lyngsø, A. Shahinyan, J. S. Pedersen, D. Otzen

{"title":"Molecular dynamics study of ACBP denaturation in alkyl sulfates demonstrates possible pathways of unfolding through fused surfactant clusters.","authors":"A. Poghosyan, Nicholas P. Schafer, Jeppe Lyngsø, A. Shahinyan, J. S. Pedersen, D. Otzen","doi":"10.1093/protein/gzz037","DOIUrl":"https://doi.org/10.1093/protein/gzz037","url":null,"abstract":"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 to more rapid denaturation, we studied denaturation using both the medium-length alkyl chain surfactant sodium dodecyl sulfate (SDS) and the long alkyl chain surfactant sodium hexadecyl sulfate (SHS). In silico denaturation on the microsecond timescale was not achieved using preformed surfactant micelles but required ACBP to be exposed to monomeric surfactant molecules. Micellar self-assembly occurred together with protein denaturation. To validate our analyses, we calculated small-angle X-ray scattering spectra of snapshots from the simulations. These agreed well with experimental equilibrium spectra recorded on ACBP-SDS mixtures with similar compositions. Protein denaturation occurs through the binding of partial micelles to multiple preferred binding sites followed by the accretion of surfactant monomers until these partial micelles merge to form a mature micelle and the protein chain is left disordered on the surface of the micelle. While the two surfactants attack in a similar fashion, SHS's longer alkyl chain leads to a more efficient denaturation through the formation of larger clusters that attack ACBP, a more rapid drop in native contacts, a greater expansion in size, as well as a more thorough rearrangement of hydrogen bonds and disruption of helices.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"52 1","pages":""},"PeriodicalIF":2.4,"publicationDate":"2019-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75185115","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: 11

User-defined single pot mutagenesis using unamplified oligo pools. 用户自定义单锅诱变使用未扩增寡核苷酸池。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-09-10 DOI: 10.1093/protein/gzz013

Angélica V Medina-Cucurella, Paul J Steiner, Matthew S Faber, Jesús Beltrán, Alexandra N Borelli, Monica B Kirby, Sean R Cutler, Timothy A Whitehead

引用次数: 17