Bioconjugate technique for site-specific attachment of IgG onto nanoparticles

将 IgG 定点附着到纳米粒子上的生物共轭技术

• 批准号: 8822592
• 负责人: Andrew Tsourkas
• 金额: $ 24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-26 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822592
• 关键词: Affinity; Affinity Chromatography; Amino Acids; Antibodies; Azides; Binding; Biological Assay; Chemistry; Complex; Copper; Cysteine; Exhibits; Exposure to; Goals; Immunoassay; Immunoglobulin G; Label; Ligands; Ligation; Link; Mutagenesis; Pharmaceutical Preparations; Production; Protein Engineering; Proteins; Recombinant Proteins; Reproducibility; Sampling; Site; Site-Directed Mutagenesis; Specificity; Surface; Techniques; Technology; Therapeutic Human Experimentation; Time; Ultraviolet Rays; antibody conjugate; cost; cost effective; crosslink; fluorophore; improved; nanoparticle; plasma protein Z; protein expression; protein purification; public health relevance; screening

DESCRIPTION (provided by applicant): Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques (e.g. EDC-NHS) exhibit low crosslinking efficiencies, hinder functionality due to non-site-specific labeling, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging and time consuming. This can result in high costs, heteregeneous samples, and poor reproducibility. To overcome these limitations, we will recombinantly express Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA will be activated and form a covalent link between the Protein Z and the bound Fc region of IgG. This technology will be combined with expressed protein ligation (EPL), which will allow for the introduction of a fluorophore and click compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This will enable crosslinked- Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzycyclooctyne-modified microplates and nanoparticles, via copper-free click chemistry. This approach is cost-effective, easily scalable, and utilizes protein production techniques that are commercially viable. Site-specific labeling has not only been shown to improve the activity of various surface-bound antibody conjugates (e.g. immunoassays and nanoparticles) but also non-surface bound antibody conjugates (e.g. antibody-drug conjugates) and thus the approach proposed here is expected to have broad applicability. The specific aims for the proposal are (1) Optimize the yield of photoreactive Protein Z expression and the efficiency of expressed protein ligation; (2) Optimize the photo-crosslinking efficiency between Protein Z and various IgG subtypes.

描述（由申请人提供）：抗体，最常见的是igg，由于其广泛的靶标，高特异性和已证实的功效，已被广泛用作研究和治疗应用的靶向配体。许多这些应用需要将抗体偶联到表面（例如纳米颗粒和微孔板）；然而，大多数传统的生物偶联技术（如EDC-NHS）表现出低交联效率，由于非位点特异性标记而阻碍功能，和/或需要蛋白质工程（如半胱氨酸把手），这在技术上具有挑战性且耗时。这可能导致成本高、样品不均匀和再现性差。为了克服这些限制，我们将重组表达结合IgG Fc区的蛋白Z，并在其结合区域内含有一个具有紫外线活性的非天然氨基酸苯甲酰苯丙氨酸（BPA）。在长波长紫外线照射下，BPA会被激活，并在蛋白Z和IgG结合的Fc区之间形成共价键。这项技术将与表达蛋白连接（EPL）相结合，这将允许引入荧光团和点击兼容