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

Structure- and sequence-based design of synthetic single-domain antibody libraries. 基于结构和序列的合成单域抗体文库设计。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa028

Alexander M Sevy, Ming-Tang Chen, Michelle Castor, Tyler Sylvia, Harini Krishnamurthy, Andrii Ishchenko, Chung-Ming Hsieh

{"title":"Structure- and sequence-based design of synthetic single-domain antibody libraries.","authors":"Alexander M Sevy, Ming-Tang Chen, Michelle Castor, Tyler Sylvia, Harini Krishnamurthy, Andrii Ishchenko, Chung-Ming Hsieh","doi":"10.1093/protein/gzaa028","DOIUrl":"https://doi.org/10.1093/protein/gzaa028","url":null,"abstract":"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, which can be costly and time-consuming. Here we describe the development of a synthetic VHH library for in vitro selection of single domain binders. We combine structure-based design and next-generation sequencing analysis to build a library with characteristics that closely mimic the natural repertoire. To validate the performance of our synthetic library, we isolated VHH against three model antigens (soluble mouse PD-1 ectodomain, amyloid-β peptide, and MrgX1 GPCR) of different sizes and characteristics. We were able to isolate diverse binders targeting different epitopes with high affinity (as high as 5 nM) against all three targets. We then show that anti-mPD-1 binders have functional activity in a receptor blocking assay.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa028","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38737808","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

Engineering a fluorescence biosensor for the herbicide glyphosate. 设计除草剂草甘膦的荧光生物传感器。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa021

Pierre-Emmanuel Y N'Guetta, Maggie M Fink, Shahir S Rizk

引用次数: 1

Engineering therapeutic antibodies for patient safety: tackling the immunogenicity problem. 为患者安全设计治疗性抗体:解决免疫原性问题。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa025

Michael Ulitzka, Stefania Carrara, Julius Grzeschik, Henri Kornmann, Björn Hock, Harald Kolmar

引用次数: 10

Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1. 利用过氧化物荧光传感器 roGFP2-Orp1 开发用于筛选氧化酶活性的细胞结合检测系统。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa019

P L Herzog, E Borghi, M W Traxlmayr, C Obinger, H D Sikes, C K Peterbauer

{"title":"Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1.","authors":"P L Herzog, E Borghi, M W Traxlmayr, C Obinger, H D Sikes, C K Peterbauer","doi":"10.1093/protein/gzaa019","DOIUrl":"10.1093/protein/gzaa019","url":null,"abstract":"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 increased throughput, the screening of significantly larger sequence spaces in acceptable time frames is achieved but usually comes at the cost of restricted applicability. In this work, we tackle this problem by utilizing roGFP2-Orp1 as a fluorescent one-component detection system for enzymatic H2O2 formation. We determined the kinetic parameters of the roGFP2-Orp1 reaction with H2O2 and established an efficient immobilization technique for the sensor on Saccharomyces cerevisiae cells employing the lectin Concanavalin A. This allowed to realize a peroxide-sensing shell on enzyme-displaying cells, a system that was successfully employed to screen for H2O2 formation of enzyme variants in a whole-cell setting.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/5c/a7/gzaa019.PMC7720637.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38478436","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: 0

Production of a novel heterodimeric two-chain insulin-Fc fusion protein. 一种新型异二聚体双链胰岛素- fc融合蛋白的产生。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa026

Christine Faust, Christian Ochs, Marcus Korn, Ulrich Werner, Jennifer Jung, Werner Dittrich, Werner Schiebler, Rolf Schauder, Ercole Rao, Thomas Langer

{"title":"Production of a novel heterodimeric two-chain insulin-Fc fusion protein.","authors":"Christine Faust, Christian Ochs, Marcus Korn, Ulrich Werner, Jennifer Jung, Werner Dittrich, Werner Schiebler, Rolf Schauder, Ercole Rao, Thomas Langer","doi":"10.1093/protein/gzaa026","DOIUrl":"https://doi.org/10.1093/protein/gzaa026","url":null,"abstract":"Insulin is a peptide hormone produced by the pancreas. The physiological role of insulin is the regulation of glucose metabolism. Under certain pathological conditions the insulin levels can be reduced leading to the metabolic disorder diabetes mellitus (DM). For type 1 DM and, dependent on the disease progression for type 2 DM, insulin substitution becomes indispensable. To relieve insulin substitution therapy for patients, novel insulin analogs with pharmacokinetic and pharmacodynamic profiles aiming for long-lasting or fast-acting insulins have been developed. The next step in the evolution of novel insulins should be insulin analogs with a time action profile beyond 1-2 days, preferable up to 1 week. Nowadays, insulin is produced in a recombinant manner. This approach facilitates the design and production of further insulin-analogs or insulin-fusion proteins. The usage of the Fc-domain from immunoglobulin as a fusion partner for therapeutic proteins and peptides is widely used to extend their plasma half-life. Insulin consists of two chains, the A- and B-chain, which are connected by two disulfide-bridges. To produce a novel kind of Fc-fusion protein we have fused the A-chain as well as the B-chain to Fc-fragments containing either 'knob' or 'hole' mutations. The 'knob-into-hole' technique is frequently used to force heterodimerization of the Fc-domain. Using this approach, we were able to produce different variants of two-chain-insulin-Fc-protein (tcI-Fc-protein) variants. The tcI-Fc-fusion variants retained activity as shown in in vitro assays. Finally, prolonged blood glucose lowering activity was demonstrated in normoglycemic rats. Overall, we describe here the production of novel insulin-Fc-fusion proteins with prolonged times of action.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa026","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38671398","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: 5

A novel phage display vector for selection of target-specific peptides. 一种新的噬菌体展示载体，用于选择目标特异性肽。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa023

Alex Chang, Joey P Ting, Alfonso Espada, Howard Broughton, Manuel Molina-Martin, Sepideh Afshar

引用次数: 4

The N-terminal 1-55 residues domain of pyruvate dehydrogenase from Escherichia coli assembles as a dimer in solution. 大肠杆菌丙酮酸脱氢酶n端1-55残基结构域在溶液中组装成二聚体。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz044

Yuanyuan Wang, Zemao Gong, Han Fang, Dongming Zhi, Hu Tao

引用次数: 0

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity. 保留功能活性的低免疫原性人源抗cd25单克隆抗体的设计、制备和体外测试

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa017

Marcia Stickler, Anita Reddy, Joanna M Xiong, Melanie H Wong, Yoshiko Akamatsu, Paul R Hinton, Fiona A Harding

{"title":"Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity.","authors":"Marcia Stickler, Anita Reddy, Joanna M Xiong, Melanie H Wong, Yoshiko Akamatsu, Paul R Hinton, Fiona A Harding","doi":"10.1093/protein/gzaa017","DOIUrl":"https://doi.org/10.1093/protein/gzaa017","url":null,"abstract":"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 by identifying and modifying a CD4+ T cell epitope region in the VH region. Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa017","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38213005","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

Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V. 暴露于一个隐蔽的Hsp70结合位点决定了als相关sod1突变体A4V的细胞毒性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa008

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

{"title":"Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V.","authors":"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","doi":"10.1093/protein/gzaa008","DOIUrl":"https://doi.org/10.1093/protein/gzaa008","url":null,"abstract":"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 sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37927125","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

Nanobody stability engineering by employing the ΔTm shift; a comparison with apparent rate constants of heat-induced aggregation. 利用ΔTm位移的纳米体稳定性工程热诱导聚合的表观速率常数的比较。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz017

Patrick Kunz, A. Ortale, N. Mücke, Katinka Zinner, J. Hoheisel

{"title":"Nanobody stability engineering by employing the ΔTm shift; a comparison with apparent rate constants of heat-induced aggregation.","authors":"Patrick Kunz, A. Ortale, N. Mücke, Katinka Zinner, J. Hoheisel","doi":"10.1093/protein/gzz017","DOIUrl":"https://doi.org/10.1093/protein/gzz017","url":null,"abstract":"The antigen-binding domains of camelid heavy-chain antibodies, also called nanobodies, gained strong attention because of their unique functional and biophysical properties. They gave rise to an entire spectrum of applications in biotechnology, research and medicine. Despite several reports about reversibly refolding nanobodies, protein aggregation plays a major role in nanobody thermoresistance, asking for strategies to engineer their refolding behavior. Here, we use measurements of nanobody aggregation kinetics to validate structural features in the nanobody fold that are suppressing heat-induced nanobody aggregation. Furthermore, the kinetic measurements yielded a detailed insight into the concept of the ΔTm shift, a metric for protein aggregation propensities obtained from differential scanning fluorimetry measurements. By relating the equilibrium measurements of the ΔTm shift to the kinetic measurements of heat-induced nanobody aggregation, a distinct relationship could be identified that allows a prediction of nanobody aggregation rates from a simple equilibrium measurement of ΔTm.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86060422","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: 8