Protein Engineering Design & Selection最新文献_第7页

Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants. 准确、高效的基于结构的计算诱变法模拟维多利亚绿荧光蛋白突变体的荧光水平。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa022

Majid Masso

{"title":"Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants.","authors":"Majid Masso","doi":"10.1093/protein/gzaa022","DOIUrl":"https://doi.org/10.1093/protein/gzaa022","url":null,"abstract":"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 compatibility relative to native GFP, as well as a vector of environmental perturbation (EP) scores characterizing the impact at all GFP residue positions. A significant GFP structure-function relationship (P < 0.0001) was elucidated by comparing the sequence-structure compatibility scores with the functional data. Next, the computed vectors for GFP mutants were used to train predictive models of fluorescence by implementing random forest (RF) classification and tree regression machine learning algorithms. Classification performance reached 0.93 for sensitivity, 0.91 for precision and 0.90 for balanced accuracy, and regression models led to Pearson's correlation as high as r = 0.83 between experimental and predicted GFP mutant fluorescence. An RF model trained on a subset of over 1000 experimental single residue GFP mutants with measured fluorescence was used for predicting the 3300 remaining unstudied single residue mutants, with results complementing known GFP biochemical and biophysical properties. In addition, models trained on the subset of experimental GFP mutants harboring multiple residue replacements successfully predicted fluorescence of the single residue GFP mutants. The models developed for this study were accurate and efficient, and their predictions outperformed those of several related state-of-the-art methods.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"33 ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38478437","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: 1

Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design. 通过合理设计提高烟曲霉纤维生物水解酶(Cel6A)的催化活性和稳定性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa020

Subba Reddy Dodda, Nibedita Sarkar, Piyush Jain, Kaustav Aikat, Sudit S Mukhopadhyay

{"title":"Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design.","authors":"Subba Reddy Dodda, Nibedita Sarkar, Piyush Jain, Kaustav Aikat, Sudit S Mukhopadhyay","doi":"10.1093/protein/gzaa020","DOIUrl":"https://doi.org/10.1093/protein/gzaa020","url":null,"abstract":"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 from Aspergillus fumigatus (AfCel6A). Structure-based variants of AfCel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5-7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"33 ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38478434","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

Protein Engineering, Design and Selection. 蛋白质工程，设计与选择。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa024

R. Chica

引用次数: 16

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid. 基于arac的生物传感器对三乙酸内酯和茴香酸的工程灵敏度和特异性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa027

Zhiqing Wang, Aarti Doshi, Ratul Chowdhury, Yixi Wang, Costas D Maranas, Patrick C Cirino

{"title":"Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid.","authors":"Zhiqing Wang, Aarti Doshi, Ratul Chowdhury, Yixi Wang, Costas D Maranas, Patrick C Cirino","doi":"10.1093/protein/gzaa027","DOIUrl":"https://doi.org/10.1093/protein/gzaa027","url":null,"abstract":"We previously described the design of triacetic acid lactone (TAL) biosensor 'AraC-TAL1', based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a 'decoy' ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"33 ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa027","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38626956","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

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":"33 ","pages":""},"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

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":"33 ","pages":""},"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

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

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

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":"33 ","pages":""},"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