Protein Engineering Design & Selection最新文献

Linker minimization and characterization of fc-fused interleukin-17A for increased in vivo half-life. fc融合白细胞介素- 17a体内半衰期增加的连接子最小化和表征。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-07-08 DOI: 10.1093/protein/gzaf009

Qiu C Wu, Jungmin Lee, Aishwarya Swaminathan, Ashley Winward, Yung Hwang, Merav Socolovsky, Jeffrey C Way, Allon M Klein

{"title":"Linker minimization and characterization of fc-fused interleukin-17A for increased in vivo half-life.","authors":"Qiu C Wu, Jungmin Lee, Aishwarya Swaminathan, Ashley Winward, Yung Hwang, Merav Socolovsky, Jeffrey C Way, Allon M Klein","doi":"10.1093/protein/gzaf009","DOIUrl":"https://doi.org/10.1093/protein/gzaf009","url":null,"abstract":"Interleukin-17A (IL-17A) is a cytokine involved in pro-inflammatory responses and tissue regeneration, with potential therapeutic and research applications. However, its short serum half-life limits in vivo use. Here, we report the systematic design of Fc-IL-17A fusion proteins for extended half-life. Through computational analysis of 25 design variants using AlphaFold, we found that IL-17A's native N-terminal unstructured region functions as a crucial natural linker that cannot be effectively replaced by artificial sequences. We therefore generated mouse and human Fc-IL-17A variants using direct N-terminal fusion without additional linkers. The resulting proteins retain IL-17A's ability to stimulate IL-6 production and erythroid cell growth. Pharmacokinetic analysis confirms that the Fc fusion increases the serum half-life in mice from 1.5 to 13 hours post-subcutaneous injection. This enables tractable experimental use of IL-17A in vivo for studying its role in inflammation and tissue repair. We further perform pharmacokinetics and pharmacodynamics modeling and propose a dosing regimen with reduced frequency of injection for delivering comparable IL-17A activity. This work provides a valuable pharmacological tool for injectable delivery, enabling investigation of IL-17A's biological functions in homeostasis and disease and exploration of its therapeutic potential in tissue regeneration.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585630","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

Dissection of the MeCP2 Repressor Protein Enables CRISPR Platform Optimization via Localization Engineering. MeCP2抑制蛋白的解剖通过定位工程实现CRISPR平台优化。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-07-02 DOI: 10.1093/protein/gzaf008

Andrew J Kristof, Krithika Karunakaran, Yann Ferry, Sophie Briggs, Christopher Allen, Paula Mizote, Zixin Jian, Costas Arvanitis, John Blazeck

{"title":"Dissection of the MeCP2 Repressor Protein Enables CRISPR Platform Optimization via Localization Engineering.","authors":"Andrew J Kristof, Krithika Karunakaran, Yann Ferry, Sophie Briggs, Christopher Allen, Paula Mizote, Zixin Jian, Costas Arvanitis, John Blazeck","doi":"10.1093/protein/gzaf008","DOIUrl":"https://doi.org/10.1093/protein/gzaf008","url":null,"abstract":"Clustered regularly interspaced short palindromic repeat interference (CRISPRi), the fusion of nuclease-inactive Cas9 with transcriptional repressor domains, is a powerful platform enabling site-specific gene knockdown across diverse biological contexts. Previously described CRISPRi systems typically utilize two distinct domain classes: (1) Krüppel-associated box domains and (2) truncations of the multifunctional protein, MeCP2. Despite widespread adoption of MeCP2 truncations for developing CRISPRi platforms, individual contributions of subdomains within MeCP2's transcriptional repression domain (TRD) toward enhancing gene knockdown remain unclear. Here, we dissect MeCP2's TRD and observe that two subdomains, the expected NcoR/SMRT interaction domain (NID) and an embedded nuclear localization signal (NLS), can separately enhance gold-standard CRISPRi platform performance beyond levels attained with the canonical MeCP2 protein truncation. Incorporating side-by-side analyses of nuclear localization and gene knockdown for over 30 constructs featuring MeCP2 subdomains or virus-derived NLS sequences, we demonstrate that appending C-terminal NLS motifs to dCas9-based transcriptional regulators, both repressors and activators, can significantly improve their effector function across several cell lines. We also observe that NLS placement greatly impacts CRISPRi repressor performance, and that modifying the subdomain configuration natively found within MeCP2 can also enhance gene suppression capabilities in certain contexts. Overall, this work demonstrates the interplay of two complimentary chimeric protein design considerations, transcriptional domain 'dissection' and NLS motif placement, for optimizing CRISPR-mediated transcriptional regulation in mammalian systems.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144546210","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

Antibody numbering schemes: advances, comparisons and tools for antibody engineering. 抗体编号方案：抗体工程的进展、比较和工具。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzaf005

Zirui Zhu, Hossein Ashrafian, Navid Mohammadian Tabrizi, Emily Matas, Louisa Girard, Haowei Ma, Edouard C Nice

引用次数: 0

A facile yeast-display approach for antibody mask discovery. 一种简单的酵母展示方法用于抗体掩膜的发现。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzaf006

Nithya M Badarinath, Basudeb Mondal, Christopher M Yellman, Kendreze L Holland, Hee Jun Lee, Hathaichanok Phuengkham, Andrew P Cazier, Jaewoo Son, Jacob R Smith, John R Cox, Andrew J Kristof, Yusef A Haikal, Gabriel A Kwong, John Blazeck

{"title":"A facile yeast-display approach for antibody mask discovery.","authors":"Nithya M Badarinath, Basudeb Mondal, Christopher M Yellman, Kendreze L Holland, Hee Jun Lee, Hathaichanok Phuengkham, Andrew P Cazier, Jaewoo Son, Jacob R Smith, John R Cox, Andrew J Kristof, Yusef A Haikal, Gabriel A Kwong, John Blazeck","doi":"10.1093/protein/gzaf006","DOIUrl":"10.1093/protein/gzaf006","url":null,"abstract":"Tuning in vivo activity of protein therapeutics can improve their safety. In this vein, it is possible to add a 'mask' moiety to a protein therapeutic such that its ability to bind its target is prevented until the mask has been proteolytically removed, for instance by a tumor-associated protease. As such, new methods to isolate functional masking sequences can aid development of protein therapies. Here, we describe a yeast display-based method to discover peptide sequences that prevent binding of antibody fragments to their antigen target. Our method includes an in situ ability to screen for restoration of binding by scFvs after proteolytic mask removal, and it takes advantage of the antigenic target itself to guide mask discovery. First, we genetically linked a yeast-displayed αPSCA scFv to overlapping 'tiles' of its target. By selecting for reduced antigen binding via flow cytometry, we discovered two peptide masks that we confirmed to be linear epitopes of the PSCA antigen. We then expanded our method towards developing masks for three-dimensional epitopes by using a co-crystal structure of an αHer2 antibody in complex with its antigen to guide combinatorial mask design. In sum, our efforts show the feasibility of employing yeast-displayed, antigen-based libraries to find antibody masks.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12123510/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144095737","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

CDRxAbs: antibody small-molecule conjugates with computationally designed target-binding synergy. CDRxAbs：具有计算设计的靶向结合协同作用的抗体小分子偶联物。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzaf004

Jingzhou Wang, Aiden J Aceves, Nicholas J Friesenhahn, Stephen L Mayo

{"title":"CDRxAbs: antibody small-molecule conjugates with computationally designed target-binding synergy.","authors":"Jingzhou Wang, Aiden J Aceves, Nicholas J Friesenhahn, Stephen L Mayo","doi":"10.1093/protein/gzaf004","DOIUrl":"10.1093/protein/gzaf004","url":null,"abstract":"Bioconjugates as therapeutic modalities combine the advantages and offset the disadvantages of their constituent parts to achieve a refined spectrum of action. We combine the concept of bioconjugation with the full atomic simulation capability of computational protein design to define a new class of molecular recognition agents: CDR-extended antibodies, abbreviated as CDRxAbs. A CDRxAb incorporates a covalently attached small molecule into an antibody/target binding interface using computational protein design to create an antibody small-molecule conjugate that binds tighter to the target of the small molecule than the small molecule would alone. CDRxAbs are also expected to increase the target binding specificity of their associated small molecules. In a proof-of-concept study using monomeric streptavidin/biotin pairs at either a nanomolar or micromolar-level initial affinity, we designed nanobody-biotin conjugates that exhibited >20-fold affinity improvement against their protein targets with step-wise optimization of binding kinetics and overall protein stability. The workflow explored through this process promises a novel approach to optimize small-molecule based therapeutics and to explore new chemical and target space for molecular-recognition agents in general.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143671679","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

Engineering of a lysosomal-targeted GAA enzyme. 溶酶体靶向GAA酶的工程设计。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzaf001

Nicholas Marze, Ilya Tikh, Susan Benard, Yuxing Cheng, Vincent Yu, Waijiao Cai, Edward Lavallie, Erin Lopez, Jing Wang, Tatyana Zamkovaya, Suryanarayan Somanathan

引用次数: 0

Tuning ProteinMPNN to reduce protein visibility via MHC Class I through direct preference optimization. 通过直接偏好优化，调整ProteinMPNN通过MHC I类降低蛋白质可见性。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzaf003

Hans-Christof Gasser, Diego A Oyarzún, Javier Antonio Alfaro, Ajitha Rajan

引用次数: 0

Engineering, design and selection of fluorescence-activating proteins for advanced imaging and biosensing. 工程，设计和选择荧光激活蛋白的先进成像和生物传感。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzaf007

Lina El Hajji, Arnaud Gautier

引用次数: 0

Optimized single-cell gates for yeast display screening. 优化单细胞门酵母显示筛选。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzae018

Xiaoli Pan, Matheus O de Souza, Francisco M Figueiras, Aric Huang, Bailey B Banach, Jacy R Wolfe, Azady Pirhanov, Bharat Madan, Brandon J DeKosky

引用次数: 0

Enhancing the Thermostability and solubility of a single-domain catalytic antibody. 提高单结构域催化抗体的热稳定性和溶解度。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2025-01-10 DOI: 10.1093/protein/gzaf002

Yunhang Cui, Xuchen Zhou, Sainan Li, Jingfei Chen, Mingming Qin, Liaoyuan An, Yefei Wang, Lishan Yao

{"title":"Enhancing the Thermostability and solubility of a single-domain catalytic antibody.","authors":"Yunhang Cui, Xuchen Zhou, Sainan Li, Jingfei Chen, Mingming Qin, Liaoyuan An, Yefei Wang, Lishan Yao","doi":"10.1093/protein/gzaf002","DOIUrl":"10.1093/protein/gzaf002","url":null,"abstract":"Catalytic antibodies have the ability to bind to and degrade antigens, offering a significant potential for therapeutic use. The light chain of an antibody, UA15-L, can cleave the peptide bond of Helicobacter pylori urease, thus inhibiting the spread of the bacteria. However, the variable domain of UA15-L has a poor thermostability and solubility. In this study, we employed a combined computational and experimental approach to enhance the protein's stability and solubility properties. The protein unfolding hotspots were initially identified using molecular dynamics simulations. Following this, a disulfide bond was designed in an unfolding hotspot to stabilize the protein. Subsequently, protein solubility was enhanced with the assistance of computational methods by introducing polar or charged residues on the protein surface. The combination of multiple mutations resulted in UA15-L variable domain variants with improved thermostability, solubility, expression, and enhanced activity at elevated temperatures. These variants represent promising candidates for further engineering of catalytic activity and specificity.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442797","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