Protein Engineering Design & Selection最新文献

{"title":"Correction to: Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIaR131 and FcγRIIaH131.","authors":"","doi":"10.1093/protein/gzag018","DOIUrl":"10.1093/protein/gzag018","url":null,"abstract":"","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"39 ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13369955/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148450383","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}

{"title":"Free energy-guided Q302P mutation in GH1 β-glucosidase from Aspergillus fumigatus improves enzyme function via catalytic tunnel perturbation.","authors":"Shalini Das, Subba Reddy Dodda, Musaddique Hossain, Soumyadeep Ray, Sudit S Mukhopadhyay","doi":"10.1093/protein/gzag015","DOIUrl":"10.1093/protein/gzag015","url":null,"abstract":"<p><p>Beta-glucosidase (EC 3.2.1.21) is crucial to biorefinery industries because it is the bottleneck enzyme in the enzymatic conversion of cellobiose to glucose, a key component of the enzyme complex that converts lignocellulosic biomass into glucose. The enzyme is inhibited by its own product, glucose, which accumulates and blocks the catalytic tunnel or the product exit region. This is the main drawback of this enzyme. In this work, we engineered AfBgl1 to enhance its thermal stability. FireProt was used to generate a mutant library, and the mutants with the highest free energies (Q302P, S187W, and S301D) were selected for further studies. The comparative analyses revealed that the mutant protein Q302P showed increased substrate affinity (Km of Q302P = 0.3 mM and AfBgl1 = 1.42 mM), activity-based thermostability (up to 70°C), and increased glucose tolerance (Ki of Q302P = 188.65 mM and AfBgl1 = 83.96 mM). Interestingly, a mutation based on free energy gains these properties of glucose tolerance and catalytic efficiency, which is generally unconventional. The mutational effect has been justified and explained through structural analysis and molecular dynamics simulations, indicating that the mutation's position is crucial for triggering this robust effect. The study delineates the scope of free-energy-based protein engineering beyond thermostability and highlights the influence of local fluctuations on the dynamics of the catalytic tunnel.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148414040","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}

{"title":"Targeted mutagenesis of a thermophilic mevalonate kinase for efficient catalysis at lower temperatures.","authors":"Sylvia A Sarnik, Jeffrey N Law, Sam J B Mallinson, Hunter B Harrington, Joel L Kaar, Daniel K Schwartz, Yannick J Bomble","doi":"10.1093/protein/gzag027","DOIUrl":"10.1093/protein/gzag027","url":null,"abstract":"<p><p>Thermophile-derived enzymes are of interest for cell-free biocatalysis due to their intrinsic thermostability, which simplifies purification via heat treatment and correlates with enhanced activity retention under cell-free reaction conditions. However, thermal stability often comes at the expense of slower turnover rates for reactions that need to be conducted at ambient temperature out of concern for cofactor stability and to reduce energy requirements. This study demonstrates a targeted engineering strategy to increase the room temperature activity of the thermophilic mevalonate kinase from Methanocaldococcus jannaschii. Site-saturation mutagenesis of evolutionarily variable residues within the mevalonate binding pocket generated numerous mutants with increased activity at 22°C. Fifteen mutants demonstrated a two- to four-fold increase in activity, and the most active mutant, I10S, achieved a five-fold increase in kcat relative to the wild-type. Importantly, the I10S mutant retained thermostability comparable to the wild-type, and a minimal decline in long-term stability was observed. This mutagenesis strategy may have broader applications towards tuning thermophilic enzyme activity at desired operating temperatures to enhance the efficiency of cell-free reactions while reducing operating costs.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13654298/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149076709","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}

{"title":"Incorporation of azide groups into an engineered Fn3 scaffold protein for site-specific, bioorthogonal conjugation.","authors":"Kristine T Le, Tatum R McKenna, Pauline M Mallari, Sheher-Bano Z Ahmed, Alexis M Ziemba, Maren E Buck, Sarah J Moore","doi":"10.1093/protein/gzag022","DOIUrl":"10.1093/protein/gzag022","url":null,"abstract":"<p><p>Protein bioconjugates have a wide range of applications, including for targeted drug delivery and in vivo diagnostics. Expanding the chemistry available to link protein to cargo remains a critical consideration for the development of novel bioconjugates for unmet needs. Currently, most FDA-approved protein bioconjugates are based on the antibody structure, which constrains the available conjugation chemistries and applications. Alternative scaffold proteins have potential as targeting molecules that advance the synthetic possibilities. Here, we report two methods of high-yield incorporation of azide-containing groups into an Fn3 domain protein scaffold to enable click chemistry conjugation. One approach incorporates noncanonical amino acid azidohomoalanine during recombinant protein expression, and the other method develops a linker system using a unique thiol residue in the Fn3 structure. These synthetic approaches enabled orthogonal, dual labeling. The resulting conjugates retain functionality in receptor binding assays, validating these methods for diverse applications in engineering scaffold proteins for bioconjugate applications.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13480472/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148698262","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}

{"title":"The Dibasics of M13 phage display: examining the substrate specificity of Escherichia coli outer membrane proteases.","authors":"Michael D Scholle, Brian K Kay","doi":"10.1093/protein/gzag023","DOIUrl":"10.1093/protein/gzag023","url":null,"abstract":"<p><p>Bacteriophage M13 substrate libraries are a useful tool to discover novel protease substrates. In this study, we have mapped 7-mer protease substrates cleaved by native Escherichia coli proteases during phage outgrowth. Cleavage was monitored by the loss of a biotinylated AviTag at the N-terminus of the recombinant M13 minor coat protein, protein III. Using this method, we isolated 120 phage-displayed substrates that contain dibasic amino acid motifs (Lys-Lys, Arg-Arg, Lys-Arg, and Arg-Lys), of which the Lys-Lys motif has not been previously described using substrate phage. Since dibasic motifs are known to be substrates of the outer membrane protein proteases OmpP and OmpT, the phage library was transferred into a mutant strain deficient in both protease activities, and the selection process was repeated. Sequencing of recovered clones revealed no enrichment of the four dibasic motifs, demonstrating that all four motifs are substrates of Omptin proteases. These findings indicate that phage propagation in Omptin-expressing bacteria leads to the loss of dibasic amino acid motifs from phage-displayed combinatorial peptide, protein, and antibody libraries. This suggests that the presence of dibasic residues in proteins and peptides displayed on bacteriophage M13 should necessitate the use of OmpT- and OmpP-deficient bacterial hosts.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148801911","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}

{"title":"Combining bacterial display and protein language models to engineer a CD69-binding affibody for molecular imaging of immune activation.","authors":"Hugo Olsson, Cornelia Westerberg, Jonas Persson, Lina Löfstrand, Olle Korsgren, Olof Eriksson, Stefan Ståhl, John Löfblom","doi":"10.1093/protein/gzag013","DOIUrl":"10.1093/protein/gzag013","url":null,"abstract":"<p><p>Recent years have seen remarkable clinical success with therapies that harness the patient's immune system, with checkpoint inhibitors in oncology as a prominent example. Non-invasive monitoring of immune activation in vivo has the potential to accelerate both basic immunology research and clinical drug development, offering a valuable means to track responses to emerging immunotherapies. CD69 is a rapidly induced activation marker on lymphocytes and other leukocytes, making it an attractive imaging target. For such applications, affibody molecules offer distinct advantages as radio imaging tracers due to their small size, high affinity, and rapid pharmacokinetics, resulting in excellent imaging contrast. Here, we combined directed evolution with computational design to optimise a CD69-binding affibody molecule. An alanine scan of the parental binder informed the construction of a diversification library, which was displayed on Escherichia coli and subjected to iterative MACS and FACS selections with stringent off-rate competition. The top selection hit was subsequently refined through site-directed mutagenesis, including variants suggested by a general protein language model. The resulting lead, Z1525, bound human CD69 with single-digit nanomolar affinity and showed improved thermal stability while retaining solubility and refolding capacity, consistent with suitability for radiolabelling and in vivo targeting. Most importantly, it displayed selective binding to CD69 on stimulated Jurkat cells, with negligible binding to resting cells. These results establish E. coli display with off-rate-driven selection as an efficient strategy for affibody affinity maturation and demonstrate that protein language models can effectively guide improvements in folding stability. Z1525 represents a promising radio imaging tracer candidate for monitoring immune activation in vivo and warrants further preclinical development.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13284772/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148220928","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}

{"title":"Assessing structural prediction accuracy for nanobody-small molecule complexes.","authors":"Berta Bori-Bru, Juan-Pablo Salvador, Ramon Crehuet","doi":"10.1093/protein/gzag021","DOIUrl":"10.1093/protein/gzag021","url":null,"abstract":"<p><p>Generative models have transformed structural bioinformatics, enabling antibody and nanobody design against protein epitopes; however, nanobody engineering for small molecule sensing remains largely experimental. In this work, we evaluate seven state-of-the-art structure predictors, AlphaFold3, Chai-1, Boltz-2x, RoseTTAFold3, Protenix, FlowDock and OmegaFold, on nanobody-small molecule complexes. Most predictors accurately reproduced nanobody and CDR geometries but struggled with ligand placement and orientation, although co-folding improved overall accuracy. Contact analysis revealed that CDR1, rather than CDR3, was predominantly involved in ligand binding. Intrinsic confidence scores correlated poorly with experimental accuracy and showed limited power in distinguishing binders from non-binders. Increasing the number of samples and seeds yielded modest gains in accuracy, whereas additional recycles did not. These findings highlight both the strengths and limitations of structure prediction methods for nanobody-small molecule complexes.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13480469/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148686769","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}

{"title":"Protein engineering: status report.","authors":"Hui-Wang Ai, Frances H Arnold, Doug Barrick, Hagan Bayley, Alfie-Louise R Brownless, Robert E Campbell, Roger Castells-Graells, Pimchai Chaiyen, Roberto A Chica, Lauren O Chisholm, Daniel Christ, Guo-Chao Chu, Michael Chungyoun, Vincent G H Eijsink, Michael B Elowitz, Douglas J Fansher, Rudi Fasan, Gabriel Foley, Michelle S Frei, Andreas Gagsteiger, Ruiyan Gao, Elizabeth M J Gillam, Jeffrey J Gray, Luis F Guerra, Birte Höcker, Zhiyang Jin, Shina Caroline Lynn Kamerlin, Nobuyasu Koga, Roland E Kontermann, Brian Kuhlman, Olivia A Langlois, Dongyang Li, Roberto D Lins, Chang C Liu, Lei Liu, Diego López Barreiro, Peilong Lu, Sohum Mehta, Ikumi Miyazaki, Manuel M Müller, Serge Muyldermans, Naoto Nemoto, Jeff Nivala, James K Nuñez, Izaiah J Ornelas, Joelle N Pelletier, Andreas Plückthun, Owen T Porth, Kridsadakorn Prakinee, Yujia Qing, Julia C Reisenbauer, Sandy Schmidt, Oliver Seifert, Mikhail G Shapiro, Rachna Sharma, Nilmani Singh, Benjamin J Smith, Jamie B Spangler, Rie Tatsumi-Koga, Takuya Terai, Henrik Terholsen, Mattias Tolhurst, Katherine W Tripp, Vanessa Tanja Trossmann, Chandra L Tucker, Onur Turak, James A Van Deventer, Anikó Várnai, Isabelle F T Viana, Amy M Weeks, K Dane Wittrup, Kingsley L-J Wong, Chenggang Xi, Todd O Yeates, Andy Hsien-Wei Yeh, Jin Zhang, Tiger S Zhang, Huimin Zhao, Fang Zheng, Jingyi Zhu, Sacha Zinn","doi":"10.1093/protein/gzag020","DOIUrl":"10.1093/protein/gzag020","url":null,"abstract":"<p><p>With this status report, we aim to provide a timely snapshot of the protein engineering field as a broad and rapidly advancing discipline that integrates computational, molecular biology, structure-guided, evolutionary, and synthetic approaches to create new and improved proteins with tailored structures and useful functions. The report is organized into eight thematic areas spanning core methodologies and major application domains, including enzymes, therapeutics, detection, synthetic biology, and materials. Contributions from experts across these areas highlight both the historical foundations and recent advances in their respective fields, with particular emphasis on the growing influence of machine learning and artificial intelligence-based methods. Emerging from this broad overview is a central message: protein engineering appears to be entering a golden age, defined by a rapidly accelerating pace of progress, even as significant challenges in design, screening, and real-world application remain. Looking ahead, the continued integration of computational and experimental strategies is poised to further accelerate the impact of protein engineering across an expanding range of economically and societally important sectors, from therapeutics and molecular imaging to diagnostics, plastic recycling, and industrial chemistry.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13500061/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148474224","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}

{"title":"Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs.","authors":"Anna Mestre Borras, Hanna Mehari, Stefan Ståhl, John Löfblom","doi":"10.1093/protein/gzag010","DOIUrl":"10.1093/protein/gzag010","url":null,"abstract":"<p><p>Antibody prodrugs provide a strategy to reduce systemic toxicity by masking therapeutic antibodies until activation in specific physiological environments. Nivolumab, an anti-PD-1 checkpoint inhibitor used in cancer immunotherapy, can cause immune-related adverse events. As a first step toward a prodrug version of nivolumab, we screened an Escherichia coli affibody library using MACS and FACS, identifying affibodies that effectively mask its PD-1-binding regions. Deep sequencing revealed an unexpected enrichment of proline-rich variants, possibly mimicking a PD-1 loop. AlphaFold modeling suggested that these affibodies form interactions with nivolumab despite low alpha-helical content. Biosensor assays confirmed effective masking in a nivolumab prodrug format, with PD-1 binding restored upon proteolytic cleavage. These findings support further exploration of PD-1-mimicking affibodies as masking domains, advancing the development of more selective nivolumab prodrugs with improved safety profiles.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13122598/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147693818","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}

{"title":"SARS-CoV-2 Orf3a protein interaction mapping using unnatural amino acid incorporation.","authors":"Eryn Lundrigan, Noreen Ahmed, John Paul Pezacki","doi":"10.1093/protein/gzag024","DOIUrl":"10.1093/protein/gzag024","url":null,"abstract":"<p><p>Mapping transient protein-protein interactions remain a major challenge in studying viral host-pathogen interfaces. While some virus-host interactions are stable and readily captured, the majority are highly dynamic, reflecting the need for viral proteins to engage distinct host factors at different stages of the life cycle. Here, we employ a protein engineering strategy based on the site-specific incorporation of the unnatural acid p-azido-L-phenylalanine (AzF) to enable photo-crosslinking proteomic analysis of the SARS-CoV-2 accessory protein Orf3a in live cells. Genetic installation of AzF at residue K198 of Orf3a permitted UV-induced covalent capture of proximal host interacting proteins, overcoming challenges associated with membrane localization and limited protein abundance. A total of 248 high-confidence Orf3a-interacting proteins were reproducibly identified and subjected to gene ontology analysis, revealing enrichment in innate immune signaling, antiviral defense, RNA processing, and viral replication-associated pathways. Orf3a is an accessory protein that functions as a viroporin and traffics across multiple cellular compartments, and was found to interact with host RNA helicases, RNA-binding proteins, immune regulators, and metabolic enzymes implicated in SARS-CoV-2 infection. Together, these results demonstrate that genetically encoded, site-specific photo-crosslinking enables selective capture of transient interactions that are often missed by nonspecific 254 nm UV crosslinking approaches and highlights Orf3a as a multifunctional protein that engages diverse host pathways. More broadly, this study establishes a generalizable framework for leveraging unnatural amino acid-based protein engineering approaches to interrogate dynamic host-pathogen interactions.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13544855/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148842291","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}