Protein Engineering Design & Selection最新文献_第4页

Engineering Candida boidinii formate dehydrogenase for activity with the non-canonical cofactor 3'-NADP(H). 利用非经典辅因子3'-NADP（H）对博伊迪尼假丝酵母甲酸脱氢酶进行活性改造。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad009

Salomon Vainstein, Scott Banta

{"title":"Engineering Candida boidinii formate dehydrogenase for activity with the non-canonical cofactor 3'-NADP(H).","authors":"Salomon Vainstein, Scott Banta","doi":"10.1093/protein/gzad009","DOIUrl":"10.1093/protein/gzad009","url":null,"abstract":"Oxidoreductases catalyze essential redox reactions, and many require a diffusible cofactor for electron transport, such as NAD(H). Non-canonical cofactor analogs have been explored as a means to create enzymatic reactions that operate orthogonally to existing metabolism. Here, we aimed to engineer the formate dehydrogenase from Candid boidinii (CbFDH) for activity with the non-canonical cofactor nicotinamide adenine dinucleotide 3'-phosphate (3'-NADP(H)). We used PyRosetta, the Cofactor Specificity Reversal Structural Analysis and Library Design (CSR-SALAD), and structure-guided saturation mutagenesis to identify mutations that enable CbFDH to use 3'-NADP+. Two single mutants, D195A and D195G, had the highest activities with 3'-NADP+, while the double mutant D195G/Y196S exhibited the highest cofactor selectivity reversal behavior. Steady state kinetic analyses were performed; the D195A mutant exhibited the highest KTS value with 3'-NADP+. This work compares the utility of computational approaches for cofactor specificity engineering while demonstrating the engineering of an important enzyme for novel non-canonical cofactor selectivity.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10146296","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

Masked inverse folding with sequence transfer for protein representation learning. 用于蛋白质表示学习的带序列转移的掩模反向折叠。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad015

Kevin K Yang, Niccolò Zanichelli, Hugh Yeh

引用次数: 0

Engineering of a phosphotriesterase with improved stability and enhanced activity for detoxification of the pesticide metabolite malaoxon. 一种具有改进的稳定性和增强的农药代谢产物马拉氧酮解毒活性的磷酸三酯酶的工程。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad020

Laura Job, Anja Köhler, Mauricio Testanera, Benjamin Escher, Franz Worek, Arne Skerra

{"title":"Engineering of a phosphotriesterase with improved stability and enhanced activity for detoxification of the pesticide metabolite malaoxon.","authors":"Laura Job, Anja Köhler, Mauricio Testanera, Benjamin Escher, Franz Worek, Arne Skerra","doi":"10.1093/protein/gzad020","DOIUrl":"10.1093/protein/gzad020","url":null,"abstract":"Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/KM >2 × 107 M-1 min-1, degradation of malaoxon is unsatisfactory (kcat/KM ≈ 1 × 104 M-1 min-1). Here, we report the rational engineering of BdPTE mutants with improved properties and their efficient production in Escherichia coli. As result, the mutant BdPTE(VRNVVLARY) exhibits 37-fold faster malaoxon hydrolysis (kcat/KM = 4.6 × 105 M-1 min-1), together with enhanced expression yield, improved thermal stability and reduced susceptibility to oxidation. Therefore, this BdPTE mutant constitutes a powerful candidate to develop a biocatalytic antidote for the detoxification of this common pesticide metabolite as well as related OP compounds.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71523393","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

Correction to: The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility. 更正：中和抗严重急性呼吸系统综合征冠状病毒2型单链抗体向IgG的可变转化提供了对RBD表位可及性的深入了解。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad018

引用次数: 0

Non-canonical amino acids as a tool for the thermal stabilization of enzymes. 作为酶热稳定工具的非典型氨基酸。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad003

Tim Lugtenburg, Alejandro Gran-Scheuch, Ivana Drienovská

引用次数: 0

A protein engineering approach toward understanding FKBP51 conformational dynamics and mechanisms of ligand binding. 一种了解FKBP51构象动力学和配体结合机制的蛋白质工程方法。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad014

Jorge A Lerma Romero, Christian Meyners, Nicole Rupp, Felix Hausch, Harald Kolmar

引用次数: 0

An easy-to-use high-throughput selection system for the discovery of recombinant protein binders from alternative scaffold libraries. 一种易于使用的高通量选择系统，用于从替代支架库中发现重组蛋白结合物。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad011

Marit Möller, Malin Jönsson, Magnus Lundqvist, Blenda Hedin, Louise Larsson, Emma Larsson, Johan Rockberg, Mathias Uhlén, Sarah Lindbo, Hanna Tegel, Sophia Hober

{"title":"An easy-to-use high-throughput selection system for the discovery of recombinant protein binders from alternative scaffold libraries.","authors":"Marit Möller, Malin Jönsson, Magnus Lundqvist, Blenda Hedin, Louise Larsson, Emma Larsson, Johan Rockberg, Mathias Uhlén, Sarah Lindbo, Hanna Tegel, Sophia Hober","doi":"10.1093/protein/gzad011","DOIUrl":"10.1093/protein/gzad011","url":null,"abstract":"Selection by phage display is a popular and widely used technique for the discovery of recombinant protein binders from large protein libraries for therapeutic use. The protein library is displayed on the surface of bacteriophages which are amplified using bacteria, preferably Escherichia coli, to enrich binders in several selection rounds. Traditionally, the so-called panning procedure during which the phages are incubated with the target protein, washed and eluted is done manually, limiting the throughput. High-throughput systems with automated panning already in use often require high-priced equipment. Moreover, the bottleneck of the selection process is usually the screening and characterization. Therefore, having a high-throughput panning procedure without a scaled screening platform does not necessarily increase the discovery rate. Here, we present an easy-to-use high-throughput selection system with automated panning using cost-efficient equipment integrated into a workflow with high-throughput sequencing and a tailored screening step using biolayer-interferometry. The workflow has been developed for selections using two recombinant libraries, ADAPT (Albumin-binding domain-derived affinity proteins) and CaRA (Calcium-regulated affinity) and has been evaluated for three new targets. The newly established semi-automated system drastically reduced the hands-on time and increased robustness while the selection outcome, when compared to manual handling, was very similar in deep sequencing analysis and generated binders in the nanomolar affinity range. The developed selection system has shown to be highly versatile and has the potential to be applied to other binding domains for the discovery of new protein binders.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/7f/95/gzad011.PMC10545973.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10278675","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

Data-driven enzyme engineering to identify function-enhancing enzymes. 数据驱动的酶工程，以识别功能增强酶。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzac009

Yaoyukun Jiang, Xinchun Ran, Zhongyue J Yang

{"title":"Data-driven enzyme engineering to identify function-enhancing enzymes.","authors":"Yaoyukun Jiang, Xinchun Ran, Zhongyue J Yang","doi":"10.1093/protein/gzac009","DOIUrl":"10.1093/protein/gzac009","url":null,"abstract":"Identifying function-enhancing enzyme variants is a 'holy grail' challenge in protein science because it will allow researchers to expand the biocatalytic toolbox for late-stage functionalization of drug-like molecules, environmental degradation of plastics and other pollutants, and medical treatment of food allergies. Data-driven strategies, including statistical modeling, machine learning, and deep learning, have largely advanced the understanding of the sequence-structure-function relationships for enzymes. They have also enhanced the capability of predicting and designing new enzymes and enzyme variants for catalyzing the transformation of new-to-nature reactions. Here, we reviewed the recent progresses of data-driven models that were applied in identifying efficiency-enhancing mutants for catalytic reactions. We also discussed existing challenges and obstacles faced by the community. Although the review is by no means comprehensive, we hope that the discussion can inform the readers about the state-of-the-art in data-driven enzyme engineering, inspiring more joint experimental-computational efforts to develop and apply data-driven modeling to innovate biocatalysts for synthetic and pharmaceutical applications.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"36 ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10365845/pdf/gzac009.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10316967","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: 3

The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility. 中和抗严重急性呼吸系统综合征冠状病毒2型单链抗体向IgG的可变转化提供了对RBD表位可及性的深入了解。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad008

Matthew R Chang, Hanzhong Ke, Laura Losada Miguéns, Christian Coherd, Katrina Nguyen, Maliwan Kamkaew, Rebecca Johnson, Nadia Storm, Anna Honko, Quan Zhu, Anthony Griffiths, Wayne A Marasco

{"title":"The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility.","authors":"Matthew R Chang, Hanzhong Ke, Laura Losada Miguéns, Christian Coherd, Katrina Nguyen, Maliwan Kamkaew, Rebecca Johnson, Nadia Storm, Anna Honko, Quan Zhu, Anthony Griffiths, Wayne A Marasco","doi":"10.1093/protein/gzad008","DOIUrl":"10.1093/protein/gzad008","url":null,"abstract":"Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe RSV infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants. Although there are many techniques for the identification of mAbs, including single B cell cloning and immunization of genetically engineered mice, the low cost, rapid throughput and technological simplicity of antibody phage display has led to its widespread adoption in mAb discovery efforts. Here we used our 27-billion-member naïve single-chain antibody (scFv) phage library to identify a panel of neutralizing anti-SARS-CoV-2 scFvs targeting diverse epitopes on the receptor binding domain (RBD). Although typically a routine process, we found that upon conversion to IgG, a number of our most potent clones failed to maintain their neutralization potency. Kinetic measurements confirmed similar affinity to the RBD; however, mechanistic studies provide evidence that the loss of neutralization is a result of structural limitations likely arising from initial choice of panning antigen. Thus this work highlights a risk of scFv-phage panning to mAb conversion and the importance of initial antigen selection.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"36 ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2023-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10505556/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10355296","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

De novo design of a polycarbonate hydrolase. 聚碳酸酯水解酶的新设计。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2023-01-21 DOI: 10.1093/protein/gzad022

Laura H Holst, Niklas G Madsen, Freja T Toftgård, Freja Rønne, Ioana-Malina Moise, Evamaria I Petersen, Peter Fojan

引用次数: 0