Protein Engineering Design & Selection最新文献_第6页

Affinity maturation of TCR-like antibodies using phage display guided by structural modeling. 在结构建模指导下利用噬菌体展示技术实现 TCR 类抗体的亲和成熟。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac005

Rahel Frick, Lene S Høydahl, Ina Hodnebrug, Erik S Vik, Bjørn Dalhus, Ludvig M Sollid, Jeffrey J Gray, Inger Sandlie, Geir Åge Løset

引用次数: 0

Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation. c端截断法降低嗜热硬脂地杆菌苹果酸脱氢酶的底物抑制作用。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac008

Yuya Shimozawa, Hinano Matsuhisa, Tsutomu Nakamura, Tomoki Himiyama, Yoshiaki Nishiya

{"title":"Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation.","authors":"Yuya Shimozawa, Hinano Matsuhisa, Tsutomu Nakamura, Tomoki Himiyama, Yoshiaki Nishiya","doi":"10.1093/protein/gzac008","DOIUrl":"https://doi.org/10.1093/protein/gzac008","url":null,"abstract":"Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate. Geobacillus stearothermophilus MDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed that C-terminal residues regulate the position of the active site loop. C-terminal deletions of gs-MDH successfully increased Ki values by 5- to 8-fold with maintained thermal stability (>90% of the wild-type enzyme), although kcat/Km values were decreased by <2-fold. The structure of the mutant showed a shift in the location of the active site loop and a decrease in its volume, suggesting that substrate inhibition was reduced by eliminating the putative substrate binding site causing inhibition. Our results provide an effective method to reduce substrate inhibition of the enzyme without loss of other parameters, including binding and stability constants.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"35 ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2022-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10384924","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

Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity. 通过蛋白质工程学方法增强单-ADP-核糖转移酶的活性检测。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac006

Albert Galera-Prat, Juho Alaviuhkola, Heli I Alanen, Lari Lehtiö

引用次数: 0

Correction to: Protease-stable DARPins as promising oral therapeutics. 更正:蛋白酶稳定的DARPins作为有前途的口服治疗药物。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac003

Rudo A Simeon, Yu Zeng, Vikas Chonira, Andrea Martinez Aguirre, Mauricio Lasagna, Marko Baloh, Joseph A Sorg, Cecilia Tommos, Zhilei Chen

引用次数: 0

Bispecific antibodies-effects of point mutations on CH3-CH3 interface stability. 点突变对 CH3-CH3 界面稳定性的影响。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac012

Nancy D Pomarici, Monica L Fernández-Quintero, Patrick K Quoika, Franz Waibl, Alexander Bujotzek, Guy Georges, Klaus R Liedl

{"title":"Bispecific antibodies-effects of point mutations on CH3-CH3 interface stability.","authors":"Nancy D Pomarici, Monica L Fernández-Quintero, Patrick K Quoika, Franz Waibl, Alexander Bujotzek, Guy Georges, Klaus R Liedl","doi":"10.1093/protein/gzac012","DOIUrl":"10.1093/protein/gzac012","url":null,"abstract":"A new format of therapeutic proteins is bispecific antibodies, in which two different heavy chains heterodimerize to obtain two different binding sites. Therefore, it is crucial to understand and optimize the third constant domain (CH3-CH3) interface to favor heterodimerization over homodimerization, and to preserve the physicochemical properties, as thermal stability. Here, we use molecular dynamics simulations to investigate the dissociation process of 19 CH3-CH3 crystal structures that differ from each other in few point mutations. We describe the dissociation of the dimeric interface as a two-steps mechanism. As confirmed by a Markov state model, apart from the bound and the dissociated state, we observe an additional intermediate state, which corresponds to an encounter complex. The analysis of the interdomain contacts reveals key residues that stabilize the interface. We expect that our results will improve the understanding of the CH3-CH3 interface interactions and thus advance the developability and design of new antibodies formats.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"35 ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2022-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9741699/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10353046","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

Stabilization of the SARS-CoV-2 receptor binding domain by protein core redesign and deep mutational scanning. 通过蛋白质核心重新设计和深度突变扫描稳定 SARS-CoV-2 受体结合域。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac002

Alison C Leonard, Jonathan J Weinstein, Paul J Steiner, Annette H Erbse, Sarel J Fleishman, Timothy A Whitehead

{"title":"Stabilization of the SARS-CoV-2 receptor binding domain by protein core redesign and deep mutational scanning.","authors":"Alison C Leonard, Jonathan J Weinstein, Paul J Steiner, Annette H Erbse, Sarel J Fleishman, Timothy A Whitehead","doi":"10.1093/protein/gzac002","DOIUrl":"10.1093/protein/gzac002","url":null,"abstract":"Stabilizing antigenic proteins as vaccine immunogens or diagnostic reagents is a stringent case of protein engineering and design as the exterior surface must maintain recognition by receptor(s) and antigen-specific antibodies at multiple distinct epitopes. This is a challenge, as stability enhancing mutations must be focused on the protein core, whereas successful computational stabilization algorithms typically select mutations at solvent-facing positions. In this study, we report the stabilization of SARS-CoV-2 Wuhan Hu-1 Spike receptor binding domain using a combination of deep mutational scanning and computational design, including the FuncLib algorithm. Our most successful design encodes I358F, Y365W, T430I, and I513L receptor binding domain mutations, maintains recognition by the receptor ACE2 and a panel of different anti-receptor binding domain monoclonal antibodies, is between 1 and 2°C more thermally stable than the original receptor binding domain using a thermal shift assay, and is less proteolytically sensitive to chymotrypsin and thermolysin than the original receptor binding domain. Our approach could be applied to the computational stabilization of a wide range of proteins without requiring detailed knowledge of active sites or binding epitopes. We envision that this strategy may be particularly powerful for cases when there are multiple or unknown binding sites.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"35 ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2022-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9077414/pdf/gzac002.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9166459","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

A multivalent antibody assembled from different building blocks using tag/catcher systems: a case study. 使用标签/捕集器系统从不同构建块组装的多价抗体:一个案例研究。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac014

Christof Schindler, Christine Faust, Hanno Sjuts, Christian Lange, Jennifer Kühn, Werner Dittrich, Wulf Dirk Leuschner, Werner Schiebler, Joachim Hofmann, Ercole Rao, Thomas Langer

{"title":"A multivalent antibody assembled from different building blocks using tag/catcher systems: a case study.","authors":"Christof Schindler, Christine Faust, Hanno Sjuts, Christian Lange, Jennifer Kühn, Werner Dittrich, Wulf Dirk Leuschner, Werner Schiebler, Joachim Hofmann, Ercole Rao, Thomas Langer","doi":"10.1093/protein/gzac014","DOIUrl":"https://doi.org/10.1093/protein/gzac014","url":null,"abstract":"The field of therapeutic antibodies and, especially bi- or multispecific antibodies, is growing rapidly. Especially for treating cancers, multispecific antibodies are very promising, as there are multiple pathways involved and multispecific antibodies offer the possibility to interfere at two or more sites. Besides being used as therapeutic, multispecific antibodies can be helpful tools in basic research. However, the design and choice of the most appropriate multispecific antibody format are far from trivial. The generation of multispecific antibodies starts with the generation of antibodies directed against the desired targets and then combining the different antigen-binding sites in one molecule. This is a time-consuming and laborious approach since the most suitable geometry cannot be predicted. The SpyTag technology is based on a split-protein system, where a small peptide of said protein, the SpyTag, can bind to the remaining protein, the SpyCatcher. An irreversible isopeptide bond between the SpyTag and the SpyCatcher is formed. A related Tag-Catcher system is the SnoopTag-SnoopCatcher. These systems offer the opportunity to separately produce proteins fused to the tag-peptides and to the catcher-domains and assemble them in vitro. Our goal was to design and produce different antibody fragments, Fab domains and Fc-containing domains, with different tags and/or catchers as building blocks for the assembly of different multivalent antibodies. We have shown that large multivalent antibodies consisting of up to seven building blocks can be prepared. Binding experiments demonstrated that all binding sites in such a large molecule retained their accessibility to their corresponding antigens.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"35 ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2022-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9226656","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

Structure-based engineering of minimal proline dehydrogenase domains for inhibitor discovery. 基于结构工程的最小脯氨酸脱氢酶结构域抑制剂发现。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzac016

Alexandra N Bogner, Juan Ji, John J Tanner

{"title":"Structure-based engineering of minimal proline dehydrogenase domains for inhibitor discovery.","authors":"Alexandra N Bogner, Juan Ji, John J Tanner","doi":"10.1093/protein/gzac016","DOIUrl":"10.1093/protein/gzac016","url":null,"abstract":"Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility and the presence of domains unrelated to PRODH activity. Herein, we report the engineering of minimal PRODH domains for inhibitor discovery. The best designs contain one-third of the 1233-residue PutA from Sinorhizobium meliloti and include a linker that replaces the PutA α-domain. The minimal PRODHs exhibit near wild-type enzymatic activity and are susceptible to known inhibitors and inactivators. Crystal structures of minimal PRODHs inhibited by S-(-)-tetrahydro-2-furoic acid and 2-(furan-2-yl)acetic acid were determined at 1.23 and 1.72 Å resolution. Minimal PRODHs should be useful in chemical probe discovery.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"35 ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2022-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9801229/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10670094","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

Yeast surface display-based identification of ACE2 mutations that modulate SARS-CoV-2 spike binding across multiple mammalian species. 基于酵母表面展示的 ACE2 突变鉴定可调节多种哺乳动物的 SARS-CoV-2 穗状结合。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2022-02-17 DOI: 10.1093/protein/gzab035

Pete Heinzelman, Jonathan C Greenhalgh, Philip A Romero

引用次数: 0

Protease-stable DARPins as promising oral therapeutics. 蛋白酶稳定的DARPins作为有前途的口服治疗药物。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab028

Rudo A Simeon, Yu Zeng, Vikas Chonira, Andrea Martinez Aguirre, Mauricio Lasagna, Marko Baloh, Joseph A Sorg, Cecilia Tommos, Zhilei Chen

{"title":"Protease-stable DARPins as promising oral therapeutics.","authors":"Rudo A Simeon, Yu Zeng, Vikas Chonira, Andrea Martinez Aguirre, Mauricio Lasagna, Marko Baloh, Joseph A Sorg, Cecilia Tommos, Zhilei Chen","doi":"10.1093/protein/gzab028","DOIUrl":"10.1093/protein/gzab028","url":null,"abstract":"Clostridioides difficile is an enteric bacterium whose exotoxins, TcdA and TcdB, inactivate small GTPases within the host cells, leading to bloody diarrhea. In prior work, our group engineered a panel of potent TcdB-neutralizing designed ankyrin repeat proteins (DARPin) as oral therapeutics against C. difficile infection. However, all these DARPins are highly susceptible to digestion by gut-resident proteases, i.e. trypsin and chymotrypsin. Close evaluation of the protein sequence revealed a large abundance of positively charged and aromatic residues in the DARPin scaffold. In this study, we significantly improved the protease stability of one of the DARPins, 1.4E, via protein engineering. Unlike 1.4E, whose anti-TcdB EC50 increased >83-fold after 1-hour incubation with trypsin (1 mg/ml) or chymotrypsin (0.5 mg/ml), the best progenies-T10-2 and T10b-exhibit similar anti-TcdB potency as their parent in PBS regardless of protease treatment. The superior protease stability of T10-2 and T10b is attributed to the removal of nearly all positively charged and aromatic residues except those directly engaged in target binding. Furthermore, T10-2 was found to retain significant toxin-neutralization ability in ex vivo cecum fluid and can be easily detected in mouse fecal samples upon oral administration. Both T10-2 and T10b enjoy a high thermo- and chemo-stability and can be expressed very efficiently in Escherichia coli (>100 mg/l in shaker flasks). We believe that, in additional to their potential as oral therapeutics against C. difficile infection, T10-2 and T10b can also serve as a new generation DARPin scaffold with superior protease stability.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"34 ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8861517/pdf/gzab028.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10431620","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