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Protein Engineering Design & Selection最新文献

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Engineering of a lysosomal-targeted GAA enzyme.
IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY 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

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.

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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 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY 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

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.

酵母展示技术在抗体发现和蛋白质工程中有着广泛的应用。酵母的细胞大小使荧光激活细胞分选(FACS)能够精确筛选基因文库,包括蛋白质表型的多参数选择。然而,酵母细胞比哺乳动物细胞表现出更广泛的大小分布,这使得单细胞门测定变得复杂。在本报告中,我们详细分析了几种酵母显示门控选项,并提出了一种通过流式细胞术选择单个酵母细胞的优化策略。这些数据揭示了优化的单细胞门控策略,以支持稳健和高效的酵母展示研究。
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引用次数: 0
Enhancing the Thermostability and solubility of a single-domain catalytic antibody.
IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY 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

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.

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引用次数: 0
Correction to: Growing ecosystem of deep learning methods for modeling protein-protein interactions. 更正:用于蛋白质-蛋白质相互作用建模的深度学习方法生态系统日益壮大。
IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-29 DOI: 10.1093/protein/gzae016
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引用次数: 0
Correction to: Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly. 更正为可变重型-可变轻型结构域和带电荷残基交换的 Fab 臂 CrossMabs,以执行正确的轻链组装。
IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-29 DOI: 10.1093/protein/gzae017
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引用次数: 0
Computational methods for protein design. 蛋白质设计计算方法
IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-29 DOI: 10.1093/protein/gzae011
Noelia Ferruz, Amelie Stein
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引用次数: 0
An engineered NKp46 antibody for construction of multi-specific NK cell engagers. 用于构建多特异性 NK 细胞吞噬体的工程化 NKp46 抗体。
IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-29 DOI: 10.1093/protein/gzae013
Robert B Lee, Sainiteesh Maddineni, Madeleine Landry, Celeste Diaz, Aanya Tashfeen, Sean A Yamada-Hunter, Crystal L Mackall, Corinne Beinat, John B Sunwoo, Jennifer R Cochran

Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity.

癌症免疫疗法的最新发展突显了利用自然杀伤(NK)细胞治疗肿瘤恶性肿瘤的潜力。其中,双特异性抗体和NK细胞吞噬蛋白疗法尤其受到关注。在这里,我们利用噬菌体展示和酵母表面展示技术设计出了一种独特的交叉反应抗体RLN131,它能与人类、小鼠和犬科动物的NKp46结合,NKp46是NK细胞上的一种激活受体。RLN131 能诱导原代 NK 细胞的增殖和活化,并被用于制造不同构型和效价的双特异性 NCKE 构合物。与未修饰的抗 CD20 单克隆抗体相比,所有 NCKE 都能增强 NK 细胞对肿瘤细胞的细胞毒性,而且无论构建物是否含有功能性 Fc 结构域,都能观察到其活性。竞争结合和精细表位图谱研究证明,RLN131 与 NKp46 上的保守表位结合,是其物种交叉反应性的基础。
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引用次数: 0
Design of functional intrinsically disordered proteins. 设计功能性本征无序蛋白。
IF 2.4 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-29 DOI: 10.1093/protein/gzae004
Ankush Garg, Nicolas S González-Foutel, Maciej B Gielnik, Magnus Kjaergaard

Many proteins do not fold into a fixed three-dimensional structure, but rather function in a highly disordered state. These intrinsically disordered proteins pose a unique challenge to protein engineering and design: How can proteins be designed de novo if not by tailoring their structure? Here, we will review the nascent field of design of intrinsically disordered proteins with focus on applications in biotechnology and medicine. The design goals should not necessarily be the same as for de novo design of folded proteins as disordered proteins have unique functional strengths and limitations. We focus on functions where intrinsically disordered proteins are uniquely suited including disordered linkers, desiccation chaperones, sensors of the chemical environment, delivery of pharmaceuticals, and constituents of biomolecular condensates. Design of functional intrinsically disordered proteins relies on a combination of computational tools and heuristics gleaned from sequence-function studies. There are few cases where intrinsically disordered proteins have made it into industrial applications. However, we argue that disordered proteins can perform many roles currently performed by organic polymers, and that these proteins might be more designable due to their modularity.

许多蛋白质并不折叠成固定的三维结构,而是在高度无序的状态下发挥作用。这些本质上无序的蛋白质给蛋白质工程和设计带来了独特的挑战:如果不对蛋白质的结构进行定制,如何才能从头开始设计蛋白质呢?在这里,我们将回顾本征无序蛋白设计这一新兴领域,重点关注其在生物技术和医学中的应用。设计目标不一定与从头设计折叠蛋白的目标相同,因为无序蛋白具有独特的功能优势和局限性。我们将重点放在本征无序蛋白具有独特功能的领域,包括无序连接体、干燥伴侣、化学环境传感器、药物输送以及生物分子凝聚物成分。功能性本征无序蛋白的设计依赖于计算工具与序列功能研究启发式方法的结合。本征无序蛋白进入工业应用的案例很少。不过,我们认为,无序蛋白质可以扮演许多目前由有机聚合物扮演的角色,而且由于其模块性,这些蛋白质可能更易于设计。
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引用次数: 0
Sequence-activity mapping via depletion reveals striking mutational tolerance and elucidates functional motifs in Tur1a antimicrobial peptide. 通过损耗绘制的序列-活性图揭示了惊人的突变耐受性,并阐明了 Tur1a 抗菌肽中的功能基团。
IF 2.4 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-29 DOI: 10.1093/protein/gzae006
Jonathan Collins, Benjamin J Hackel

Proline-rich antimicrobial peptides (PrAMPs) are attractive antibiotic candidates that target gram-negative bacteria ribosomes. We elucidated the sequence-function landscape of 43 000 variants of a recently discovered family member, Tur1a, using the validated SAMP-Dep platform that measures intracellular AMP potency in a high-throughput manner via self-depletion of the cellular host. The platform exhibited high replicate reproducibility (ρ = 0.81) and correlation between synonymous genetic variants (R2 = 0.93). Only two segments within Tur1a exhibited stringent mutational requirements to sustain potency: residues 9YLP11 and 19FP20. This includes the aromatic residue in the hypothesized binding domain but not the PRP domain. Along with unexpected mutational tolerance of PRP, the data contrast hypothesized importance of the 1RRIR4 motif and arginines in general. In addition to mutational tolerance of residue segments with presumed significance, 77% of mutations are functionally neutral. Multimutant performance mainly shows compounding effects from removed combinations of prolines and arginines in addition to the two segments of residues showing individual importance. Several variants identified as active from SAMP-Dep were externally produced and maintained activity when applied to susceptible species exogenously.

富脯氨酸抗菌肽(PrAMPs)是针对革兰氏阴性细菌核糖体的有吸引力的候选抗生素。我们利用经过验证的 SAMP-Dep 平台阐明了最近发现的 Tur1a 家族成员的 43000 个变体的序列功能图谱,该平台通过细胞宿主的自我耗竭以高通量方式测量细胞内 AMP 的效力。该平台具有很高的重复再现性(ρ = 0.81)和同义遗传变异之间的相关性(R2 = 0.93)。Tur1a 中只有两个区段显示出维持效力的严格突变要求:残基 9YLP11 和 19FP20。这包括假定结合结构域中的芳香残基,但不包括 PRP 结构域。除了 PRP 的突变耐受性出乎意料之外,数据还对比了 1RRIR4 矩阵和一般精氨酸的假设重要性。除了具有假定重要性的残基段的突变耐受性外,77% 的突变在功能上是中性的。多突变表现主要显示了除显示出单独重要性的两段残基外,去除的脯氨酸和精氨酸组合所产生的复合效应。从 SAMP-Dep 中鉴定出的几种具有活性的变异体是外源生产的,当外源应用于易感物种时仍保持活性。
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引用次数: 0
Sequence-developability mapping of affibody and fibronectin paratopes via library-scale variant characterization. 通过文库规模的变体特征描述,绘制亲和素和纤连蛋白旁位点的序列-可发展性图谱。
IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-29 DOI: 10.1093/protein/gzae010
Gregory H Nielsen, Zachary D Schmitz, Benjamin J Hackel

Protein developability is requisite for use in therapeutic, diagnostic, or industrial applications. Many developability assays are low throughput, which limits their utility to the later stages of protein discovery and evolution. Recent approaches enable experimental or computational assessment of many more variants, yet the breadth of applicability across protein families and developability metrics is uncertain. Here, three library-scale assays-on-yeast protease, split green fluorescent protein (GFP), and non-specific binding-were evaluated for their ability to predict two key developability outcomes (thermal stability and recombinant expression) for the small protein scaffolds affibody and fibronectin. The assays' predictive capabilities were assessed via both linear correlation and machine learning models trained on the library-scale assay data. The on-yeast protease assay is highly predictive of thermal stability for both scaffolds, and the split-GFP assay is informative of affibody thermal stability and expression. The library-scale data was used to map sequence-developability landscapes for affibody and fibronectin binding paratopes, which guides future design of variants and libraries.

蛋白质显影性是用于治疗、诊断或工业应用的必要条件。许多可开发性测定的通量都很低,这就限制了它们在蛋白质发现和进化后期阶段的应用。最近的方法可以对更多的变体进行实验或计算评估,但对不同蛋白质家族和可开发性指标的适用范围还不确定。在这里,我们评估了三种库规模的检测方法--酵母蛋白酶、分裂绿色荧光蛋白(GFP)和非特异性结合--预测小型蛋白质支架 affibody 和纤维连接蛋白的两个关键可开发性结果(热稳定性和重组表达)的能力。这些检测方法的预测能力是通过线性相关模型和在库规模检测数据上训练的机器学习模型进行评估的。酵母上蛋白酶检测对两种支架的热稳定性都有很高的预测性,而分裂-GFP 检测则能提供 affibody 热稳定性和表达的信息。文库规模数据被用于绘制亲和体和纤连蛋白结合旁位体的序列可发展性图谱,为未来的变体和文库设计提供指导。
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Protein Engineering Design & Selection
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