Protein Engineering Design & Selection最新文献

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.

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.

Pompe disease is a tissue glycogen disorder caused by genetic insufficiency of the GAA enzyme. GAA enzyme replacement therapies for Pompe disease have been limited by poor lysosomal trafficking of the recombinant GAA molecule through the native mannose-6-phosphate-mediated pathway. Here, we describe the successful rational engineering of a chimeric GAA enzyme that utilizes the binding affinity of a modified IGF-II moiety to its native receptor to bypass the mannose-6-phosphate-mediated lysosomal trafficking pathway, conferring a significant increase in cellular uptake of the GAA enzyme. We also demonstrate the ablation of binding between our modified IGF-II tag and two off-target receptors: IGF-I receptor and insulin receptor, as well as preserved enzymatic activity of the chimeric GAA molecule.

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.

ProteinMPNN is widely used in protein design workflows due to its ability to identify amino acid sequences that fold into specific 3D protein structures. In our work, we adjust ProteinMPNN to design proteins for a given 3D protein structure with reduced immune-visibility to cytotoxic T lymphocytes that recognize proteins via the MHC-I pathway. To achieve this, we developed a novel framework that integrates direct preference optimization (DPO)-a tuning method originally designed for large language models-with MHC-I peptide presentation predictions. This approach fosters the generation of designs with fewer MHC-I epitopes while preserving the protein's original structure. Our results demonstrate that DPO effectively reduces MHC-I visibility without compromising the structural integrity of the proteins.

Engineering improved protease activity using directed evolution is challenged by uncertainty in sequence-function mapping and inefficiency in evaluating activity of candidate mutants. We implemented a generalizable yeast surface display approach that co-displays protease mutants with substrate on the same Aga2 anchor protein. Identification of enhanced activity mutants is enabled by protease cleavage of tethered substrate removing an N-terminal epitope tag, which empowers flow cytometric isolation of cells with a decrease in signal from fluorophore-linked anti-epitope antibodies. The sequence space of tobacco etch virus protease (TEVp), commonly used for specific cleavage of recombinant protein affinity tags, has previously been investigated through random mutagenesis. Leveraging our display platform, we performed high throughput screens on seven active site combinatorial libraries created via saturation mutagenesis. Beneficial mutations were incorporated into a single second-generation library, which was screened to identify individual beneficial mutations that performed optimally in a multi-mutant context. The vast majority of resultant TEVp multi-mutants improved catalytic efficiency, generally by decreasing KM. The yeast surface protease/substrate co-display system, the insights gleaned on rational library design and mutation combination strategy, and the TEVp sequence-function map will aid future protease engineering efforts.

Fluorescence-activating proteins (FAP) have emerged as a novel class of genetically encoded tools for fluorescence-based protein imaging, complementing the existing toolkit consisting of fluorescent proteins and self-labeling tags. FAP have the ability to bind and activate the fluorescence of small molecules, called fluorogens, that are otherwise non-fluorescent, allowing protein localization with high specificity and little background. In this review, we present the engineering of FAP and FAP-based reporters from various protein scaffolds, focusing on the different strategies implemented to design and engineer their properties for specific biological imaging applications.

Erythropoietin (EPO) suppresses apoptosis and promotes survival by signaling through EPO-R/EPO-R on hematopoietic progenitors or EPO-R/CD131 on non-hematopoietic cells. However, EPO signaling through EPO-R/CD131 is controversial and there is no solved structure of a complex. Here, we constructed a structural model of EPO-R/CD131 and designed several anti-EPO-R, anti-CD131 bispecific proteins that selectively activate EPO-R/CD131. Treatment with these fusion proteins is sufficient to activate STAT5 phosphorylation downstream of EPO-R/CD131 without engaging EPO-R/EPO-R. We demonstrated that proteins with a tandem scFv or bispecific antibody format activate EPO-R/CD131, in contrast to an equimolar mixture of the individual scFvs. Finally, we explored the effect of modifications to binding domain arrangement and linker length and found results consistent with our structural model of an EPO-R/CD131 complex. These findings highlight the utility of bispecific scaffolds in the development of cytokine receptor agonists and provide a foundation for the study of EPO-R/CD131 biology and future clinical development.

Edited by: Robert E. Campbell Based on the Anticalin H1GA which tightly binds Aβ40 and Aβ42 peptides - both established biomarkers of Alzheimer's disease - we describe the design of a protein-dye conjugate as analytical reagent that shows strongly elevated fluorescence upon Aβ binding. An unpaired Cys residue was introduced at seven positions within the four loop segments that shape the ligand pocket of the engineered lipocalin. Five of these mutants were purified in the monomeric state and allowed the site-specific conjugation with IANBD amide as a solvatochromic fluorophore. Three conjugates showed ligand-dependent fluorescence and one of these, derived from H1GA(D45C), exhibited sixfold higher emission at 546 nm upon complex formation with the peptide while revealing a low KD value of 1.2 ± 0.8 nM, even in the presence of 5% (w/v) albumin. This NBD-conjugated Anticalin offers a novel biosensor with potential for the detection of Aβ peptides in biochemical assays or human body fluid samples.

Yeast display is a widely used technology in antibody discovery and protein engineering. The cell size of yeast enables fluorescence-activated cell sorting (FACS) to precisely screen gene libraries, including for multi-parameter selection of protein phenotypes. However, yeast cells show a broader size distribution than mammalian cells that complicates single-cell gate determination for FACS. In this report, we analyze several yeast display gating options in detail and present an optimized strategy to select single yeast cells via flow cytometry. These data reveal optimized single-cell gating strategies to support robust and high-efficiency yeast display studies.