Protein Engineering, Design and Selection最新文献_第4页

Rational design of glycoengineered interferon-&bgr; analogs with improved aggregation state: experimental validation 糖工程干扰素的合理设计具有改进聚合状态的类似物:实验验证

Protein Engineering, Design and Selection Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw058

M. Samoudi, Z. Minuchehr, S. Harcum, F. Tabandeh, N. Omid Yeganeh, M. Khodabandeh

{"title":"Rational design of glycoengineered interferon-&bgr; analogs with improved aggregation state: experimental validation","authors":"M. Samoudi, Z. Minuchehr, S. Harcum, F. Tabandeh, N. Omid Yeganeh, M. Khodabandeh","doi":"10.1093/protein/gzw058","DOIUrl":"https://doi.org/10.1093/protein/gzw058","url":null,"abstract":"Recombinant human interferon-&bgr; (rhIFN-&bgr;) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-&bgr; analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-&bgr; analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-&bgr; (Opt-IFN-&bgr;) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-&bgr;, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-&bgr; had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-&bgr;. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-&bgr; analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"18 1","pages":"23–30"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74684320","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: 6

Design and expression of a chimeric vaccine candidate for avian necrotic enteritis 禽坏死性肠炎候选嵌合疫苗的设计与表达

Protein Engineering, Design and Selection Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw060

A. Rostami, F. Goshadrou, R. P. Langroudi, S. Z. Bathaie, A. Riazi, J. Amani, G. Ahmadian

{"title":"Design and expression of a chimeric vaccine candidate for avian necrotic enteritis","authors":"A. Rostami, F. Goshadrou, R. P. Langroudi, S. Z. Bathaie, A. Riazi, J. Amani, G. Ahmadian","doi":"10.1093/protein/gzw060","DOIUrl":"https://doi.org/10.1093/protein/gzw060","url":null,"abstract":"Necrotic enteritis is an economically important disease of poultry mainly caused by Clostridium perfringens. The bacteria release multiple toxins of which NetB, alpha toxin and TpeL have been reported to play important roles in pathogenicity and/or severity of the disease. In this study, the sequence of clostridial toxins NetB, alpha toxin and TpeL were analyzed using bioinformatics tools to determine protein domains with high immunogenicity factor. Several chimeric trivalent proteins consisting of the immunogenic regions of the three toxins were designed and evaluated. The separate regions were fused together using rigid linkers. Based on a modeled tertiary structure, a proper combination was selected and expressed in a bacterial host (Escherichia coli) and successfully purified. The expression of the chimeric protein was further verified by western blotting. The ability of the immunized serum in recognizing each individual subunit of the chimeric protein was also examined. Circular dichroism was used to evaluate the predicted secondary structure of the chimeric protein. In vitro potency test demonstrated that the serum from a rabbit immunized with the chimeric protein is able to partially neutralize Alpha toxin, hence the construct can potentially be used as a vaccine against C. perfringens.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"6 1","pages":"39–45"},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82990690","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: 16

A robust cosolvent-compatible halohydrin dehalogenase by computational library design 基于计算库设计的稳健的助溶剂相容卤代醇脱卤酶

Protein Engineering, Design and Selection Pub Date : 2016-12-19 DOI: 10.1093/protein/gzw068

Hesam Arabnejad, Marco Dal Lago, P. Jekel, Robert J. Floor, A. Thunnissen, A. C. Terwisscha van Scheltinga, Hein J. Wijma, D. Janssen

{"title":"A robust cosolvent-compatible halohydrin dehalogenase by computational library design","authors":"Hesam Arabnejad, Marco Dal Lago, P. Jekel, Robert J. Floor, A. Thunnissen, A. C. Terwisscha van Scheltinga, Hein J. Wijma, D. Janssen","doi":"10.1093/protein/gzw068","DOIUrl":"https://doi.org/10.1093/protein/gzw068","url":null,"abstract":"To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"58 1","pages":"173–187"},"PeriodicalIF":0.0,"publicationDate":"2016-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85654055","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: 34

A theoretical study on the reactivity of the Mo/Cu-containing carbon monoxide dehydrogenase with dihydrogen 含Mo/ cu一氧化碳脱氢酶与二氢反应性的理论研究

Protein Engineering, Design and Selection Pub Date : 2016-12-19 DOI: 10.1093/protein/gzw071

R. Breglia, M. Bruschi, U. Cosentino, L. De Gioia, C. Greco, Toshiko Miyake, G. Moro

引用次数: 4

Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates 谷氨酸棒状杆菌中龙胆酸1,2-双加氧酶底物范围的扩大，用于单羟基苯甲酸盐的转化

Protein Engineering, Design and Selection Pub Date : 2016-11-24 DOI: 10.1093/protein/gzw061

E. Eppinger, A. Stolz

{"title":"Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates","authors":"E. Eppinger, A. Stolz","doi":"10.1093/protein/gzw061","DOIUrl":"https://doi.org/10.1093/protein/gzw061","url":null,"abstract":"The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the &agr;-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme (‘salicylate dioxygenase’, SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C. glutamicum ATCC 13032. The combination of sequence comparisons with previously performed structural and mutational analyses of the SDO allowed to identify an amino acid residue (Ala112) which might prevent the oxidation of (substituted) salicylate(s) by the GDO from C. glutamicum. Therefore, the relevant mutation (Ala→Gly) was introduced into the GDO from C. glutamicum. The GDO variant obtained gained the ability to oxidise salicylate and several other monohydroxylated substrates. In order to screen a broader range of enzyme variants a chromogenic assay was developed which allowed the detection of bacterial colonies converting salicylate. The applicability of this test system was proven by screening a set of GDO variants obtained by saturation mutagenesis at different positions. This demonstrated that also GDO variants carrying the mutations Ala112→Ser, Ala112→Ile and Ala112→Asp converted salicylate.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"37 1","pages":"57–65"},"PeriodicalIF":0.0,"publicationDate":"2016-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85321721","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: 6

Conformational flexibility of an anti-IL-13 DARPin† 抗il -13 DARPin†的构象柔韧性

Protein Engineering, Design and Selection Pub Date : 2016-11-23 DOI: 10.1093/protein/gzw059

A. Teplyakov, T. Malia, G. Obmolova, S. Jacobs, K. O'Neil, G. Gilliland

{"title":"Conformational flexibility of an anti-IL-13 DARPin†","authors":"A. Teplyakov, T. Malia, G. Obmolova, S. Jacobs, K. O'Neil, G. Gilliland","doi":"10.1093/protein/gzw059","DOIUrl":"https://doi.org/10.1093/protein/gzw059","url":null,"abstract":"Designed ankyrin repeat proteins (DARPin®) are artificial non-immunoglobulin binding proteins with potential applications as therapeutic molecules. DARPin 6G9 binds interleukin-13 with high affinity and blocks the signaling pathway and as such is promising for the treatment of asthma and other atopic diseases. The crystal structures of DARPin 6G9 in the unbound form and in complex with IL-13 were determined at high resolution. The DARPin competes for the same epitope as the IL-13 receptor chain 13R&agr;1 but does not interfere with the binding of the other receptor chain, IL-4R&agr;. Analysis of multiple copies of the DARPin molecule in the crystal indicates the conformational instability in the N-terminal cap that was predicted from molecular dynamics simulations. Comparison of the DARPin structures in the free state and in complex with IL-13 reveals a concerted movement of the ankyrin repeats upon binding resulted in the opening of the binding site. The induced-fit mode of binding employed by DARPin 6G9 is very unusual for DARPins since they were designed as particularly stable and rigid molecules. This finding shows that DARPins can operate by various binding mechanisms and suggests that some flexibility in the scaffold may be an advantage.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"118 1","pages":"31–37"},"PeriodicalIF":0.0,"publicationDate":"2016-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76934647","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

Understanding the role of phosphorylation in the binding mechanism of a PDZ domain 了解磷酸化在PDZ结构域结合机制中的作用

Protein Engineering, Design and Selection Pub Date : 2016-10-19 DOI: 10.1093/protein/gzw055

A. Toto, A. Mattei, P. Jemth, S. Gianni

{"title":"Understanding the role of phosphorylation in the binding mechanism of a PDZ domain","authors":"A. Toto, A. Mattei, P. Jemth, S. Gianni","doi":"10.1093/protein/gzw055","DOIUrl":"https://doi.org/10.1093/protein/gzw055","url":null,"abstract":"The PDZ domain is one of the most common protein–protein interaction domains in mammalian species. While several studies have demonstrated the importance of phosphorylation in interactions involving PDZ domains, there is a paucity of detailed mechanistic data addressing how the PDZ interaction is affected by phosphorylation. Here, we address this question by equilibrium and kinetic binding experiments using PDZ2 from protein tyrosine phosphatase L1 and its interaction with a peptide from the natural ligand RIL. The results show that phosphorylation of a serine residue in the RIL peptide has dual and opposing effects: it increases both the association and dissociation rate constants, which leads to an overall weakening of binding. Furthermore, we performed binding experiments with a RIL peptide in which the serine was replaced by a glutamate, a commonly used method to mimic phosphorylation in proteins. Strikingly, both the affinity and the ionic strength dependence of the affinity differed markedly for the phosphoserine and glutamate peptides. These results show that, in this particular case, glutamate is a poor mimic of serine phosphorylation.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"4 1","pages":"1–5"},"PeriodicalIF":0.0,"publicationDate":"2016-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79481611","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: 15

Direct determination of enzyme kinetic parameters from single reactions using a new progress curve analysis tool 用新的进展曲线分析工具直接测定单反应的酶动力学参数

Protein Engineering, Design and Selection Pub Date : 2016-10-15 DOI: 10.1093/protein/gzw053

Felix K. Bäuerle, Á. Zotter, G. Schreiber

引用次数: 14

Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation 设计一个最小的G蛋白，以促进G蛋白偶联受体在其活性构象中的结晶

Protein Engineering, Design and Selection Pub Date : 2016-09-26 DOI: 10.1093/protein/gzw049

B. Carpenter, C. Tate

{"title":"Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation","authors":"B. Carpenter, C. Tate","doi":"10.1093/protein/gzw049","DOIUrl":"https://doi.org/10.1093/protein/gzw049","url":null,"abstract":"G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs. Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"91 1","pages":"583 - 594"},"PeriodicalIF":0.0,"publicationDate":"2016-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79524110","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: 105

Overcoming a species-specificity barrier in development of an inhibitory antibody targeting a modulator of tumor stroma 在肿瘤基质调节剂的抑制抗体开发中克服物种特异性障碍

Protein Engineering, Design and Selection Pub Date : 2016-01-26 DOI: 10.1093/protein/gzv067

I. Grossman, T. Ilani, S. Fleishman, D. Fass

{"title":"Overcoming a species-specificity barrier in development of an inhibitory antibody targeting a modulator of tumor stroma","authors":"I. Grossman, T. Ilani, S. Fleishman, D. Fass","doi":"10.1093/protein/gzv067","DOIUrl":"https://doi.org/10.1093/protein/gzv067","url":null,"abstract":"The secreted disulfide catalyst Quiescin sulfhydryl oxidase-1 (QSOX1) affects extracellular matrix organization and is overexpressed in various adenocarcinomas and associated stroma. Inhibition of extracellular human QSOX1 by a monoclonal antibody decreased tumor cell migration in a cell co-culture model and hence may have therapeutic potential. However, the species specificity of the QSOX1 monoclonal antibody has been a setback in assessing its utility as an anti-metastatic agent in vivo, a common problem in the antibody therapy industry. We therefore used structurally guided engineering to expand the antibody species specificity, improving its affinity toward mouse QSOX1 by at least four orders of magnitude. A crystal structure of the re-engineered variant, complexed with its mouse antigen, revealed that the antibody accomplishes dual-species targeting through altered contacts between its heavy and light chains, plus replacement of bulky aromatics by flexible side chains and versatile water-bridged polar interactions. In parallel, we produced a surrogate antibody targeting mouse QSOX1 that exhibits a new QSOX1 inhibition mode. This set of three QSOX1 inhibitory antibodies is compatible with various mouse models for pre-clinical trials and biotechnological applications. In this study we provide insights into structural blocks to cross-reactivity and set up guideposts for successful antibody design and re-engineering.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"22 1","pages":"135 - 147"},"PeriodicalIF":0.0,"publicationDate":"2016-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88120794","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: 11