RAPID PROTEIN PURIFICATION BY POLYMER BRUSH-MODIFIED MEMBRANES

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，由于蛋白质在色谱凝胶的孔中的缓慢扩散，常常在分离过程中存在瓶颈。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快速的质量运输到结合位点。这种流动还可以有效地去除不需要的蛋白质以提高纯度。然而，膜吸收剂没有广泛用于蛋白质分离，因为它们具有低蛋白质结合能力。这项工作的目的是用功能性聚合物刷修饰膜，以增加蛋白质结合能力一个数量级，并实现快速，选择性的蛋白质纯化。此外，适当设计的刷子将抵抗非特异性吸附，并提供新的方法来纯化不适合基于柱的纯化的“粘性”蛋白质。这项研究将涉及聚合物刷改性膜的合成和表征，结合组氨酸6-和谷胱甘肽S-转移酶标记的蛋白质与最小的非特异性吸附。初步结果表明，在细胞提取物中纯化组氨酸6标记的蛋白质，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刷改性膜的聚合物支持与孔径，将允许大幅度增加渗透性。这将允许使用具有高得多的容量的较低压力和较厚的膜。这种膜的形成需要开发与聚合物支持物相容的新合成方法，以及快速结合更多蛋白质的更厚刷子的生长。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，膜组成和几何形状的函数进行检查。有了新的膜，各种标签蛋白在E。大肠杆菌中的SNAP-50、人MIP合酶和SNAPc复合物将被纯化。MIP合酶对肌醇生物合成至关重要，而SNAP蛋白对基因表达至关重要。此外，SNAP-50提供了不能用典型的亲和凝胶纯化的粘性蛋白的实例。公共卫生相关性：这项研究将产生快速，廉价的方法来分离非常纯的蛋白质。这种技术将是至关重要的生产治疗性蛋白质以及研究的目的是分离蛋白质，以了解其结构和健康相关的功能。

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.