RAPID PROTEIN PURIFICATION BY POLYMER BRUSH-MODIFIED MEMBRANES

通过聚合物刷修饰膜快速纯化蛋白质

• 批准号: 7924257
• 负责人: MERLIN L BRUENING
• 金额: $ 17.43万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924257
• 关键词: Adsorption; Affinity; Aluminum Oxide; Anabolism; Architecture; Binding; Binding Sites; Biomedical Research; Caliber; Cattle; Cell Extracts; Complex; Data; Development; Diffusion; Drops; Ensure; Escherichia coli; Future; Gel; Gene Expression; Glutathione; Glutathione S-Transferase; Growth; Hand; Health; Heart; Human; Inositol; Investigation; Ions; Literature; Membrane; Metals; Methods; Modeling; Modification; Molecular Weight; Myoglobin; Nuclear; Nylons; Permeability; Plant Resins; Polymers; Porosity; Process; Production; Protein Binding; Proteins; Radial; Reporting; Research; Resistance; S-nitro-N-acetylpenicillamine; Series; Serum; Solutions; Speed; Structure; Surface; System; Techniques; Thick; Time; Ubiquitin; Work; base; density; design; macromolecule; polymerization; pressure; professor; protein function; protein purification; public health relevance; research and development; research study; therapeutic protein

DESCRIPTION (provided by applicant): Protein purification is a vital and expensive step in biomedical research and in the development and manufacturing of therapeutic proteins. Unfortunately, affinity methods, which are at the heart of most protein purifications, often present a bottleneck in the separation process because of slow diffusion of proteins in the pores of chromatographic gels. Protein-absorbing membranes can overcome this challenge because convective flow through membrane pores provides rapid mass transport to binding sites. Such flow can also effectively remove undesired proteins to increase purity. However, membrane absorbers are not widely used for protein isolation because they have low protein-binding capacities. The aim of this work is to modify membranes with functional polymer brushes to increase protein-binding capacities by an order of magnitude and enable rapid, selective protein purification. Additionally, properly designed brushes will be resistant to nonspecific adsorption and provide new methods for purification of "sticky" proteins that are not amenable to column-based purification. This research will involve synthesis and characterization of polymer brush-modified membranes that bind histidine6- and glutathione S-transferrase-tagged proteins with minimal nonspecific adsorption. Preliminary results demonstrated purification of a histidine6-tagged protein in a cell extract, with a purity that greatly exceeds similar resin-based purification. Future work aims at developing brush-modified membranes in polymeric supports with pore sizes that will allow large increases in permeability. This will permit the use of lower pressures and thicker membranes with much higher capacities. Formation of such membranes will require both development of new synthetic methods that are compatible with polymer supports and growth of thicker brushes that rapidly bind more protein. Hence, protein binding and non-specific adsorption will be examined as a function of brush thickness, composition, density, and functionalization, and membrane composition and geometry. With new membranes in hand, a variety of tagged proteins expressed in E. coli will be purified including SNAP-50, human MIP synthase, and SNAPc complex. MIP synthase is vital for inositol biosynthesis, while SNAP proteins are critical in gene expression. Additionally, SNAP-50 provides an example of a sticky protein that cannot be purified with typical affinity gels. PUBLIC HEALTH RELEVANCE: This research will yield rapid, inexpensive methods for isolating remarkably pure proteins. Such techniques will be crucial in production of therapeutic proteins as well as research studies aimed at isolating proteins to understand their structure and health- related function.

描述（由申请人提供）：蛋白质纯化是生物医学研究以及治疗性蛋白质的开发和制造中至关重要且昂贵的步骤。不幸的是，作为大多数蛋白质纯化的核心的亲和方法经常在分离过程中出现瓶颈，因为蛋白质在色谱凝胶孔中的扩散缓慢。蛋白质吸收膜可以克服这一挑战，因为通过膜孔的对流提供了到结合位点的快速质量传输。这样的流动还可以有效去除不需要的蛋白质以提高纯度。然而，膜吸收器并未广泛用于蛋白质分离，因为它们的蛋白质结合能力较低。这项工作的目的是用功能性聚合物刷修饰膜，以将蛋白质结合能力提高一个数量级，并实现快速、选择性的蛋白质纯化。此外，正确设计的刷子将能够抵抗非特异性吸附，并为纯化不适合柱纯化的“粘性”蛋白质提供新方法。这项研究将涉及聚合物刷修饰膜的合成和表征，该膜以最小的非特异性吸附结合组氨酸 6 和谷胱甘肽 S 转移酶标记的蛋白质。初步结果表明，细胞提取物中组氨酸 6 标记的​​蛋白质得到纯化，其纯度大大超过类似的基于树脂的纯化。未来的工作旨在开发具有孔径大小的聚合物支撑体的刷改性膜，从而大幅提高渗透性。这将允许使用更低的压力和更厚的膜以及更高的容量。这种膜的形成既需要开发与聚合物支撑物兼容的新合成方法，又需要生长更厚的刷子以快速结合更多的蛋白质。因此，蛋白质结合和非特异性吸附将作为刷厚度、组成、密度和功能化以及膜组成和几何形状的函数进行检查。有了新的膜，在大肠杆菌中表达的各种标记蛋白将被纯化，包括 SNAP-50、人 MIP 合酶和 SNAPc 复合物。 MIP 合酶对于肌醇生物合成至关重要，而 SNAP 蛋白对于基因表达至关重要。此外，SNAP-50 提供了无法用典型亲和凝胶纯化的粘性蛋白的示例。公共健康相关性：这项研究将产生快速、廉价的方法来分离非常纯的蛋白质。这些技术对于治疗性蛋白质的生产以及旨在分离蛋白质以了解其结构和健康相关功能的研究至关重要。