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和治疗性蛋白质开发和制造中至关重要且昂贵的一步。不幸的是，亲和方法是大多数蛋白质纯化的核心，由于蛋白质在色谱凝胶的孔中扩散缓慢，通常在分离过程中出现瓶颈。蛋白质吸收膜可以克服这一挑战，因为通过膜孔的对流流动提供了快速的质量运输到结合位点。这种流动也可以有效地去除不需要的蛋白质，以提高纯度。然而，由于膜吸收剂结合蛋白的能力较低，膜吸收剂并没有广泛应用于蛋白质分离。这项工作的目的是用功能性聚合物刷修饰膜，以提高蛋白质结合能力的数量级，并实现快速，选择性的蛋白质纯化。此外，适当设计的刷将抵抗非特异性吸附，并为“粘性”蛋白质的纯化提供新的方法，这些蛋白质不适合柱基纯化。这项研究将包括聚合物刷修饰膜的合成和表征，该膜结合组氨酸和谷胱甘肽s -转移酶标记的蛋白质，并以最小的非特异性吸附。初步结果表明，纯化的组氨酸6标记的蛋白质在细胞提取物，纯度大大超过类似的树脂为基础的纯化。未来的工作目标是在聚合物支架中开发具有孔径的电刷改性膜，这将允许渗透率大幅增加。这将允许使用更低的压力和更厚的膜，具有更高的容量。这种膜的形成既需要开发与聚合物支架兼容的新合成方法，也需要生长更厚的能快速结合更多蛋白质的刷子。因此，蛋白质结合和非特异性吸附将作为电刷厚度、组成、密度和功能化以及膜组成和几何形状的函数进行研究。有了新的膜，大肠杆菌中表达的各种标记蛋白将被纯化，包括SNAP-50、人MIP合成酶和SNAPc复合物。MIP合成酶对肌醇的生物合成至关重要，而SNAP蛋白在基因表达中至关重要。此外，SNAP-50提供了一个不能用典型亲和凝胶纯化的粘性蛋白的例子。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。

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.