Protein Engineering Design & Selection最新文献

{"title":"Engineering a Hyperstable Synthetic Miniprotein for B7-H3 Targeting.","authors":"Hannah K Windsor, Nicholas C Ulen, Claudia Beaudry, Benjamin J Hackel","doi":"10.1093/protein/gzag031","DOIUrl":"https://doi.org/10.1093/protein/gzag031","url":null,"abstract":"<p><p>Small protein ligands, such as miniproteins, have the potential to provide the efficient physiological transport and rapid clearance required for radioligand therapy. Yet, binders require single-digit nanomolar affinity for potent targeting and high stability for radiolabeling and physiological integrity. High affinity and stable single-domain miniproteins are also modular candidates for multispecific applications in cell engagers and avid constructs. Previous work demonstrated computationally designed 40-amino acid β⍺ββ miniproteins as effective scaffolds for ligand discovery. Herein, to yield the desired single-digit nanomolar affinity for B7-H3, an important immune regulator and cell surface antigen upregulated in numerous tumors, we used structural modeling to guide experimental evolution of the lead molecule. We performed constrained diversification at six sites on a β-sheet, structurally distinct from the originally engineered ⍺-helical paratope, as well as nine sites within the ⍺-helix, and sorted for strong binding to B7-H3 via yeast display and flow cytometry with detergent-solubilized cell lysate. Top variants discovered from this campaign yielded affinities of 8.4 and 11 nM, with the most performant variant, named βm.1, resulting in 1.2 ± 0.2 nM affinity. βm.1 had four β-sheet mutations relative to parental and Tm,app ≥91 °C, with reversible unfolding. The lead variant, βm.1, putatively engages B7-H3 at the same domain I epitope as the parental lead but with improved engagement via a T27F mutation and three homologous mutations. βm.1 is a hyperstable, high affinity miniprotein B7-H3 binder with compelling potential for radioligand therapy and multispecific applications.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149247453","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":"Improved Calreticulin Nanobody by Framework Engineering.","authors":"Logan Mavar, Mikhail Pavlenok, Avraneel Paul, Lucinda Hall, Benjamin M Larimer, Michael Niederweis","doi":"10.1093/protein/gzag030","DOIUrl":"https://doi.org/10.1093/protein/gzag030","url":null,"abstract":"<p><p>Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complementarity-determining region 2 (CDR2) of a soluble, clinically validated HER2 nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered the CALR nanobody 02 by adapting the core of Cal3 to a consensus framework sequence for stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a soluble protein with an increased thermal stability and a five-fold higher calreticulin binding affinity (KD: 25 nM) compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.</p>","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2026-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149240374","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":"A photo-enhanced oxidative coupling for site-specific protein Labeling via noncanonical amino acid incorporation.","authors":"Xin Shang, Bin Ma, Wei Niu, Jiantao Guo","doi":"10.1093/protein/gzag011","DOIUrl":"10.1093/protein/gzag011","url":null,"abstract":"<p><p>Genetically encoded noncanonical amino acids (ncAAs) enable site-specific installation of chemical functionalities into proteins, expanding the scope of protein engineering and bioconjugation. Here we report a photo-enhanced oxidative coupling strategy that leverages a vinyl sulfide-containing ncAA for selective protein labeling. An ortho-naphthoquinone methide (oNQM) intermediate is generated in situ from a stable precursor under mild oxidative conditions using ferricyanide, and labeling efficiency is markedly enhanced upon 365 nm irradiation. Ethyl vinyl sulfide was identified as a compact, electronically suitable dienophile that can be incorporated into proteins either through lysine modification or via genetic encoding of N6-((2-(vinylthio)ethoxy)carbonyl)-L-lysine (VtK). Under photo-enhanced oxidative conditions, robust labeling was observed at pH 6-7 with minimal background modification of wild-type proteins. Site-specific incorporation of VtK into the outer membrane protein OmpX enabled selective labeling in cell lysates and in live Escherichia coli cells, demonstrating compatibility with complex biological environments. This work establishes a genetically encodable, photo-enhanced oxidative coupling modality that complements existing ncAA-based bioconjugation strategies and expands the protein engineering toolbox.</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/PMC13170128/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147857635","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":"Mapping functional dynamics hotspots for protein engineering with NMR peak intensity analysis.","authors":"Adam M Damry, Serena E Hunt, Sandrine Legault, Michael C Thompson, Natalie K Goto, Roberto A Chica","doi":"10.1093/protein/gzag014","DOIUrl":"10.1093/protein/gzag014","url":null,"abstract":"<p><p>Structural dynamics play a crucial role in protein function, and tuning these dynamics through mutagenesis has emerged as a promising strategy for enhancing activity. However, identifying dynamics hotspots for protein engineering remains a labor-intensive challenge. Here, we demonstrate that NMR peak intensity analysis-a rapid, qualitative method with residue-level resolution-can identify functionally relevant dynamic regions with high precision. Using a family of red fluorescent proteins (RFPs) as a case study, we reveal that flexibility in specific regions of their structures correlates with function. Specifically, as quantum yield increases, the side of the β-barrel closest to the chromophore phenolate moiety becomes more rigid, while the opposite side, closest to the acylimine group, gains flexibility. Notably, the phenolate face corresponds to a mutational hotspot frequently targeted in directed evolution campaigns aimed at enhancing brightness, underscoring its functional significance. B-factor analysis of non-cryogenic X-ray crystal structures further supports our findings. Our results establish NMR peak intensity analysis as a promising tool for mapping functional dynamics hotspots to guide protein engineering campaigns.</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/PMC13336630/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148388357","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":"Computational redesign of a thermostable T7 RNA polymerase.","authors":"Zachary T Baumer, Timothy A Whitehead","doi":"10.1093/protein/gzag008","DOIUrl":"10.1093/protein/gzag008","url":null,"abstract":"<p><p>T7 RNA polymerase (T7 RNAP) is a foundational enzyme for biotechnology, but its utility for many potential applications is limited by low thermal stability of 43-44°C. While stabilized variants exist, the most stable commercial version has a proprietary sequence. In this work we developed a highly stable T7 RNAP using structure-based computational design. We combined mutations from previous stabilized variants (M5, M8, V7abcd) with new mutations identified by PROSS. These mutations were filtered using data-driven heuristics to preserve function. Our final design, T7T+, contains 30 point mutations from the original T7 RNAP and demonstrates a functional stability (T50) of 54.9°C in a thermal challenge assay, which is 2.4°C higher than the most stable, published open-source variant to date. Circular dichroism spectroscopy showed an apparent melting temperature of 53.8°C. T7T+ retains 59% of wild-type activity at 37°C. 16 of the 18 tested protein designs had higher stability against thermal challenge compared with the genetic background, attesting to the high success rates of existing non deep learning computational methods for the design of stable, functional proteins. A plasmid encoding T7T+ has been deposited in AddGene and is freely available for non-commercial use.</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/PMC13633397/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147391485","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":"A tethered yeast surface display system facilitates enriching epitope-specific binding interactions.","authors":"Rojhae A Panton, Jakob Lind, Amanda Richardson, Isabela Panozzo, Daniela Gomez Garcia, Syed Muzaffar, Lawrence A Stern","doi":"10.1093/protein/gzag019","DOIUrl":"10.1093/protein/gzag019","url":null,"abstract":"<p><p>Engineering proteins for therapeutic applications is a field that has seen substantial growth in the past two decades, but challenges remain. A major deficiency in current strategies is the lack of a means to efficiently screen for binding to key epitopes to yield a desired function. To meet this challenge, we designed a tethered yeast surface display construct that leverages high local concentrations by tethering a candidate protein binder to a target protein of interest (POI) via a flexible peptide linker. These high local concentrations enable screening of epitope-specific binders based on decreased signal due to inhibitory function when the construct is assayed alongside a competitive POI binder. We demonstrate that epitope-specific screening and enrichment is possible based on fluorescent output and study key optimization parameters. We anticipate this technique will accelerate binder development by reducing the need for downstream low throughput epitope mapping of isolated proteins.</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/PMC13480473/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148474178","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":"Examining selection dynamics and limitations in multi-round protein selection of high diversity libraries.","authors":"John Z Chen, Barnabas Gall, Tommy Y Lu, Isabella Heslop, Daniel Hesselson, Christoph Nitsche, Wai-Hong Tham, Richard J Payne, Colin J Jackson","doi":"10.1093/protein/gzag012","DOIUrl":"10.1093/protein/gzag012","url":null,"abstract":"<p><p>Proteins and peptides underpin essential biological functions and technological applications, from targeting disease-relevant interactions to providing broad enzymatic activities. However, engineering molecules with desired properties remains difficult, owing to complex sequence-structure-function relationships and the lack of data on specific systems. Experimental selection strategies, including directed evolution, phage display, and mRNA display, address this challenge by leveraging high diversity libraries and iterative enrichment under defined selection pressures. This allows for the identification of candidates without requiring extensive prior knowledge, and can generate extensive datasets for use in machine learning. While many selection systems exist, comparisons across different selection approaches are hindered by the lack of a unifying analytical framework. Here, we developed a toolset of broadly applicable analyses for assessing selection dynamics in multi-round or multi-condition experiments, ranging from position level analysis of sequence properties to full sequence space mappings through protein language model embeddings. Performing analyses across different systems, we identify desirable traits in selection experiments including enrichment of distinct sequence patterns and correlation between enrichment and final desired functions. Notably, even under weak selection regimes with all sequences <1% frequency, functional sequences (e.g., 70nM IC50 binder to SARS-CoV-2 main protease) are still consistently enriched. We also find repeated selections of the same starting library can help differentiate selection effects of varying conditions (e.g., different delivery of metal ligand) from system noise. These findings, along with the toolset, can be used to guide experimental design, interpretation, and troubleshooting across protein and peptide discovery platforms.</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/PMC13245734/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148139915","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":"Integrating machine learning tools in protein design: a case of MHETase engineering for PET biodeconstruction.","authors":"Rommel Santiago Granja-Travez, Taraka Dale, Ramesh K Jha","doi":"10.1093/protein/gzag009","DOIUrl":"10.1093/protein/gzag009","url":null,"abstract":"<p><p>The integration of machine learning tools into protein engineering offers substantial promise, yet linking computational predictions to experimental performance remains challenging. Here, we applied accessible computational platforms to engineer Ideonella sakaiensis MHETase, a key enzyme in the biodeconstruction of polyethylene terephthalate (PET). Homologue identification with EnzymeMiner, followed by solubility-focused redesign using AggreProt and ProteinMPNN, enabled the finding of MHETase-like enzymes and the generation of is-MHETase variants with increase in soluble expression, as supported by predictive scoring and experimental validation. However, retaining catalytic activity proved considerably more difficult. Structural modeling and kinetic analyses revealed that ProteinMPNN introduced substitutions incompatible with MHETase function, reflecting biases toward sequence patterns common in broader esterase families rather than constraints specific to this narrow enzyme subgroup. These findings highlight both the utility and the current limitations of accessible ML tools for enzyme engineering, underscoring the continued necessity of experimental validation to guide and refine computational predictions.</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":"147693765","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":"Engineering isoform-selective miniprotein binders to the fetal form of the insulin receptor.","authors":"Melissa A Walsh, Paul L Blanchard, Xihong Zhang, Nathaniel T Cheung, Douglas Yee, Benjamin J Hackel","doi":"10.1093/protein/gzag025","DOIUrl":"10.1093/protein/gzag025","url":null,"abstract":"<p><p>The fetal form of the insulin receptor (IRA) is predominantly expressed in many cancers, where IR signaling promotes progression of the disease. Alternatively, the adult IR isoform (IRB) is predominantly expressed in normal tissues. Previous attempts to target IR indiscriminately via dual IR/IGF1R tyrosine kinase inhibitors frequently failed due to on-target off-tumor effects. However, an IRA-specific therapeutic has the potential to specifically target cancer while sparing normal tissues. Furthermore, an IRA-specific binder would advance research regarding this disease-associated isoform. Yet, isoform-specific IR binders have not been developed. Herein, we engineer six high-affinity, IRA-specific protein binders from a synthetic miniprotein scaffold. These miniprotein variants have IRA KDs ranging from 0.98-7.6 nM with no appreciable IRB binding at 100 nM ligand. Five of the six variants are synergistic agonists. One variant (V1) is a passive, IRA-specific binder with a KD = 6.3 nM and no IRB binding at 1 μM ligand as well as no IGF1R binding at 100 nM, which provides compelling potential as a targeted therapeutic. Additionally, we present the discovery process for these molecules, a yeast display directed evolution campaign utilizing a combination of sorting techniques. Retrospective analysis of the discovery process revealed the importance of alternating selections for target affinity and isoform specificity to generate binders with a favorable combination of these properties.</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/PMC13557182/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148882548","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 and kinetic profiling of a thrombin-activable prodrug antibody targeting P-selectin.","authors":"Mie M Mortensen, Susanne N Grell, Thomas Egebjerg, Che Yang, Nikolai Lorenzen, Peter Kristensen, Jais R Bjelke","doi":"10.1093/protein/gzag029","DOIUrl":"10.1093/protein/gzag029","url":null,"abstract":"<p><p>Antibody prodrugs represent a promising therapeutic format that provides advantages compared to conventional monoclonal antibody therapeutics. In this study, we engineered an antibody prodrug using an anti-P-selectin antibody as a model system, incorporating N-terminal heavy chain extensions comprising a linear P-selectin (CD62P) epitope for shielding, a thrombin-cleavable linker, and variable spacer in the linker. A prodrug antibody library was prepared comprising various N-terminal extensions of the heavy chain including an Ala-scan over the P-selectin epitope and the thrombin cleavage site sequence (50 variants in the library). The shielding library was thoroughly investigated for retained target binding kinetics and cleavage activation kinetics. Antibody prodrug variants achieving complete shielding of target binding to a completely open state were observed in the library (many orders of magnitude reduced binding based on surface plasmon resonance (SPR) kinetic measurement). Upon thrombin activation, the antibody prodrug variants obtained full target binding at low nM affinity. Thus, at such nM antibody prodrug concentrations, the liberated shielding extensions appeared to diffuse effectively away from the paratope upon cleavage based on SPR and fluorescence-activated cell sorting (FACS) analyses. Importantly, binding studies of variants of the linear epitope peptide alone showed many orders-of-difference in reduced binding compared to the fused extensions of the epitope sequence to the antibody prodrug. Comparison of the kinetic data with a structure model of the prodrug antibody reveals aspects of designing an optimal prodrug antibody that may have general applicability as a novel therapeutic format.</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":"149118474","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}