Protein Engineering Design & Selection最新文献

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.

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.

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.

Nanobodies offer unique advantages in biomedical and biotechnological applications due to their smaller size, ability to bind challenging epitopes, and affordable production using recombinant technology. However, challenges in large-scale production, stability, and solubility limit their widespread use. To address this, we use artificial intelligence tools to optimize the scaffold region of nanobodies. We apply our approach to four nanobodies against clinically relevant targets: the cytokine tumor necrosis factor alpha, the chemotherapeutic drug methotrexate, the pancreatic biomarker amylase, and the placental hormone chorionic gonadotropin. For all the nanobodies tested, we improve stability, production, and intracellular stability while maintaining antigen-binding affinity. Our results thus demonstrate the potential for using AI-driven protein engineering to enhance the properties of nanobodies, offering insights into the interplay between stability, solubility, and antigen binding. Given the high conservation of the scaffold, we propose some mutations that could directly transfer to other nanobodies, providing an easy-to-implement, generalizable engineering strategy.

Catechol 2,3-dioxygenases (C23DO) catalyze extradiol cleavage of catechol in aromatic hydrocarbon degradation pathways. Here, we report mutagenesis studies on two C23DO isoenzymes from Diaphorobacter sp. strain DS2, aimed at elucidating the mechanism. Docking studies with various substrates on the two isozymes identified critical active-site residues and second-sphere interactions contributing to substrate binding and catalysis. These modeling studies identified eight site-directed mutants, which were designed, produced, and kinetically characterized. Substitution of histidine-to-glutamine (His-to-Gln) (H206Q64/H200Q68) activates the previously inert substrate 2,3-dihydroxybenzoic acid (2,3-DHBA), allowing its intradiol cleavage under strong acidic conditions. The mutants retained ~20-30% of their native activity toward catechol but exhibited a mechanistic switch from Fe2+-assisted extradiol to Fe3+-mediated intradiol cleavage with 2,3-DHBA. These results highlight the catalytic adaptability of acid-base residues and demonstrate how subtle second-sphere mutation can alter dioxygenase regiospecificity, providing new insights for biocatalyst engineering and environmental bioremediation.