Protein Engineering, Design and Selection最新文献_第2页

Adaption of human antibody λ and κ light chain architectures to CDR repertoires 人抗体λ和κ轻链结构对CDR库的适应

Protein Engineering, Design and Selection Pub Date : 2019-03-01 DOI: 10.1093/protein/gzz012

R. van der Kant, Joschka Bauer, Anne R. Karow-Zwick, Sebastian Kube, P. Garidel, M. Blech, F. Rousseau, J. Schymkowitz

{"title":"Adaption of human antibody λ and κ light chain architectures to CDR repertoires","authors":"R. van der Kant, Joschka Bauer, Anne R. Karow-Zwick, Sebastian Kube, P. Garidel, M. Blech, F. Rousseau, J. Schymkowitz","doi":"10.1093/protein/gzz012","DOIUrl":"https://doi.org/10.1093/protein/gzz012","url":null,"abstract":"Abstract Monoclonal antibodies bind with high specificity to a wide range of diverse antigens, primarily mediated by their hypervariable complementarity determining regions (CDRs). The defined antigen binding loops are supported by the structurally conserved β-sandwich framework of the light chain (LC) and heavy chain (HC) variable regions. The LC genes are encoded by two separate loci, subdividing the entity of antibodies into kappa (LCκ) and lambda (LCλ) isotypes that exhibit distinct sequence and conformational preferences. In this work, a diverse set of techniques were employed including machine learning, force field analysis, statistical coupling analysis and mutual information analysis of a non-redundant antibody structure collection. Thereby, it was revealed how subtle changes between the structures of LCκ and LCλ isotypes increase the diversity of antibodies, extending the predetermined restrictions of the general antibody fold and expanding the diversity of antigen binding. Interestingly, it was found that the characteristic framework scaffolds of κ and λ are stabilized by diverse amino acid clusters that determine the interplay between the respective fold and the embedded CDR loops. In conclusion, this work reveals how antibodies use the remarkable plasticity of the beta-sandwich Ig fold to incorporate a large diversity of CDR loops.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79995913","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Engineering the cofactor specificity of an alcohol dehydrogenase via single mutations or insertions distal to the 2′-phosphate group of NADP(H) 通过单次突变或插入NADP(H)的2 ' -磷酸基远端来设计乙醇脱氢酶的辅助因子特异性

Protein Engineering, Design and Selection Pub Date : 2017-05-01 DOI: 10.1093/protein/gzx009

K. Solanki, Walaa Abdallah, S. Banta

{"title":"Engineering the cofactor specificity of an alcohol dehydrogenase via single mutations or insertions distal to the 2′-phosphate group of NADP(H)","authors":"K. Solanki, Walaa Abdallah, S. Banta","doi":"10.1093/protein/gzx009","DOIUrl":"https://doi.org/10.1093/protein/gzx009","url":null,"abstract":"There have been many reports exploring the engineering of the cofactor specificity of aldo-keto reductases (AKRs), as this class of proteins is ubiquitous and exhibits many useful activities. A common approach is the mutagenesis of amino acids involved in interactions with the 2'-phosphate group of NADP(H) in the cofactor binding pocket. We recently performed a 'loop-grafting' approach to engineer the substrate specificity of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus and we found that a loop insertion after residue 211, which is on the back side of the cofactor binding pocket, could also alter cofactor specificity. Here, we further explore this approach by introducing single point mutations and single amino acid insertions at the loop insertion site. Six different mutants of AdhD were created by either converting glycine 211 to cysteine or serine or by inserting alanine, serine, glycine or cysteine between the 211 and 212 residues. Several mutants gained activity with NADP+ above the wild-type enzyme. And remarkably, it was found that all of the mutants investigated resulted in some degree of reversal of cofactor specificity in the oxidative direction. These changes were generally a result of changes in conformations of the ternary enzyme/cofactor/substrate complexes as opposed to changes in affinities or binding energies of the cofactors. This study highlights the role that amino acids which are distal to the cofactor binding pocket but are involved in substrate interactions can influence cofactor specificity in AdhD, and this strategy should translate to other AKR family members.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87826838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 12

Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins 区域间连接子的插入提高了Zygote arrest (Zar)融合蛋白的表达和生物活性

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzx002

J. Cook, A. Charlesworth

{"title":"Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins","authors":"J. Cook, A. Charlesworth","doi":"10.1093/protein/gzx002","DOIUrl":"https://doi.org/10.1093/protein/gzx002","url":null,"abstract":"Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites; however, these fusion proteins expressed poorly and/or lacked robust function. Here, we tested the effect of inserting additional linkers between the fusion domains. Three linkers were tested, each 17 amino acids long with different physical and chemical properties: flexible hydrophilic, rigid extended or rigid helical. In the presence of any of the three linkers, GST-Zar1 and GST-Zar2 had fewer breakdown products. Moreover, in the presence of any of the linkers, MS2-Zar1 was expressed to higher levels, and in dual luciferase tethered assays, both MS2-Zar1 and MS2-Zar2 repressed luciferase translation to a greater extent. These data suggest that for Zar fusion proteins, increasing the length of linkers, regardless of their physical or chemical properties, improves stability, expression and bioactivity.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89179325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme. 羧肽酶 G2 循环排列工程，用于设计一种自动抑制酶。

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzx005

Brahm J Yachnin, Sagar D Khare

{"title":"Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme.","authors":"Brahm J Yachnin, Sagar D Khare","doi":"10.1093/protein/gzx005","DOIUrl":"10.1093/protein/gzx005","url":null,"abstract":"Carboxypeptidase G2 (CPG2) is an Food and Drug Administration (FDA)-approved enzyme drug used to treat methotrexate (MTX) toxicity in cancer patients receiving MTX treatment. It has also been used in directed enzyme-prodrug chemotherapy, but this strategy has been hampered by off-site activation of the prodrug by the circulating enzyme. The development of a tumor protease activatable CPG2, which could be achieved using a circular permutation of CPG2 fused to an inactivating 'prodomain', would aid in these applications. We report the development of a protease accessibility-based screen to identify candidate sites for circular permutation in proximity of the CPG2 active site. The resulting six circular permutants showed similar expression, structure, thermal stability, and, in four cases, activity levels compared to the wild-type enzyme. We rationalize these results based on structural models of the permutants obtained using the Rosetta software. We developed a cell growth-based selection system, and demonstrated that when fused to periplasm-directing signal peptides, one of our circular permutants confers MTX resistance in Escherichia coli with equal efficiency as the wild-type enzyme. As the permutants have similar properties to wild-type CPG2, these enzymes are promising starting points for the development of autoinhibited, protease-activatable zymogen forms of CPG2 for use in therapeutic contexts.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283397/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73471949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","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 the expression of an anti-interleukin-13 antibody through rational design and mutagenesis 通过合理设计和诱变工程表达抗白细胞介素-13抗体

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzx001

B. Popovic, Suzanne J Gibson, Tarik Senussi, Sara Carmen, S. Kidd, T. Slidel, I. Strickland, Jianqing Xu, J. Spooner, A. Lewis, N. Hudson, Lorna Mackenzie, J. Keen, B. Kemp, C. Hardman, D. Hatton, T. Wilkinson, T. Vaughan, D. Lowe

{"title":"Engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis","authors":"B. Popovic, Suzanne J Gibson, Tarik Senussi, Sara Carmen, S. Kidd, T. Slidel, I. Strickland, Jianqing Xu, J. Spooner, A. Lewis, N. Hudson, Lorna Mackenzie, J. Keen, B. Kemp, C. Hardman, D. Hatton, T. Wilkinson, T. Vaughan, D. Lowe","doi":"10.1093/protein/gzx001","DOIUrl":"https://doi.org/10.1093/protein/gzx001","url":null,"abstract":"High levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. Here, we describe two related antibodies, Ab001 and Ab008, where Ab001 shows a markedly lower level of expression relative to Ab008 when stably expressed in Chinese hamster ovary cells. We use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the VL CDR2 of Ab001. We adopted two approaches to improve the expression of Ab001. First, we used mutagenesis to change single amino-acid residues in the Ab001 VL back to the equivalent Ab008 residues but this resulted in limited improvements in expression. In contrast when we used an in silico structure-based design approach to generate a set of five individual single-point variants in a discrete region of the VL, all exhibited significantly improved expression relative to Ab001. The most successful of these, D53N, exhibited a 25-fold increase in stable transfectants relative to Ab001. The functional potency of these VL-modified antibodies was unaffected. We expect that this in silico engineering strategy can be used to improve the expression of other antibodies and proteins.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82274398","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein 通过与嗜热宿主蛋白的组合插入融合稳定环状芽孢杆菌木聚糖酶

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzw081

Vandan Shah, B. Pierre, Tamari Kirtadze, Seung-Yeol Shin, J. Kim

{"title":"Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein","authors":"Vandan Shah, B. Pierre, Tamari Kirtadze, Seung-Yeol Shin, J. Kim","doi":"10.1093/protein/gzw081","DOIUrl":"https://doi.org/10.1093/protein/gzw081","url":null,"abstract":"High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX). Specifically, we used an engineered transposon to construct a combinatorial library of randomly inserted BCX into PfMBP. The library was then subjected to functional screening to identify successful PfMBP-BCX insertion complexes, PfMBP-BCX161 and PfMBP-BCX165, displaying substantially improved kinetic stability at elevated temperatures compared to unfused BCX and other controls. Results from subsequent characterizations were consistent with the view that lowered aggregation of BCX and reduced conformational flexibility at the termini was responsible for increased thermal stability. Our stabilizing approach neither sacrificed xylanase activity nor required changes in the BCX amino acid sequence. Overall, the current study demonstrated the benefit of combinatorial insertional fusion to PfMBP as a systematic tool for the creation of enzymatically active and thermostable BCX variants.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82539105","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display 结合动物免疫和酵母展示制备人双特异性普通轻链抗体

Protein Engineering, Design and Selection Pub Date : 2017-04-01 DOI: 10.1093/protein/gzw077

Simon Krah, C. Schröter, Carla Eller, Laura Rhiel, Nicolas Rasche, J. Beck, Carolin Sellmann, Ralf Günther, L. Toleikis, B. Hock, H. Kolmar, Stefan Becker

{"title":"Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display","authors":"Simon Krah, C. Schröter, Carla Eller, Laura Rhiel, Nicolas Rasche, J. Beck, Carolin Sellmann, Ralf Günther, L. Toleikis, B. Hock, H. Kolmar, Stefan Becker","doi":"10.1093/protein/gzw077","DOIUrl":"https://doi.org/10.1093/protein/gzw077","url":null,"abstract":"Bispecific antibodies (bsAbs) pave the way for novel therapeutic modes of action along with potential benefits in several clinical applications. However, their generation remains challenging due to the necessity of correct pairings of two different heavy and light chains and related manufacturability issues. We describe a generic approach for the generation of fully human IgG-like bsAbs. For this, heavy chain repertoires from immunized transgenic rats were combined with either a randomly chosen common light chain or a light chain of an existing therapeutic antibody and screened for binders against tumor-related targets CEACAM5 and CEACAM6 by yeast surface display. bsAbs with subnanomolar affinities were identified, wherein each separate binding arm mediated specific binding to the respective antigen. Altogether, the described strategy represents a combination of in vivo immunization with an in vitro selection method, which allows for the integration of existing therapeutic antibodies into a bispecific format.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76727885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 37

Ligand-induced conformational changes in prolyl oligopeptidase: a kinetic approach 配体诱导的脯氨酰寡肽酶的构象变化:动力学方法

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzw079

R. V. Elzen, E. Schoenmakers, Inger Brandt, P. Veken, A. Lambeir

引用次数: 8

An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge. 酶门户网站的更新：探索酶知识的综合方法。

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzx008

S Pundir, J Onwubiko, R Zaru, S Rosanoff, R Antunes, M Bingley, X Watkins, C O'Donovan, M J Martin

{"title":"An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge.","authors":"S Pundir, J Onwubiko, R Zaru, S Rosanoff, R Antunes, M Bingley, X Watkins, C O'Donovan, M J Martin","doi":"10.1093/protein/gzx008","DOIUrl":"10.1093/protein/gzx008","url":null,"abstract":"14: Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al. , (2013), BMC Bioinformatics , (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al ., Nucleic Acids Res. 2016; , D385-D395) Rhea-a database of enzyme-catalysed reactions (Morgat, A., et al ., Nucleic Acids Res. 2015; , D459-D464), Reactome-a database of biochemical pathways (Fabregat, A., et al ., Nucleic Acids Res. 2016; , D481-D487), IntEnz-a resource with enzyme nomenclature information (Fleischmann, A., et al ., Nucleic Acids Res. 2004 , D434-D437) and ChEBI (Hastings, J., et al ., Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al ., Nucleic Acids Res. 2014 , 1083-1090)-resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91329337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Secretion of functional formate dehydrogenase in Pichia pastoris 毕赤酵母中功能性甲酸脱氢酶的分泌

Protein Engineering, Design and Selection Pub Date : 2017-03-01 DOI: 10.1093/protein/gzx010

M. Takacs, O. Makhlynets, Patricia L. Tolbert, I. Korendovych

引用次数: 5