RAPID PROTEIN PURIFICATION BY POLYMER BRUSH-MODIFIED MEMBRANES

描述(申请人提供)：在生物医学研究和治疗性蛋白质的开发和制造中，蛋白质纯化是一个重要且昂贵的步骤。不幸的是，作为大多数蛋白质纯化核心的亲和方法，由于蛋白质在层析凝胶的孔道中扩散缓慢，往往成为分离过程中的瓶颈。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传输到结合部位。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，由于膜吸收材料对蛋白质的结合能力较低，因此膜吸收材料在蛋白质分离中的应用并不广泛。这项工作的目的是用功能聚合物刷对膜进行修饰，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，设计合理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柱基纯化的“粘性”蛋白质。这项研究将涉及聚合物刷子改性膜的合成和表征，该膜结合组氨酸6和谷胱甘肽S转移酶标记的蛋白质，并具有最小的非特异性吸附。初步结果表明，在细胞提取液中纯化了一种组氨酸标记的蛋白质，其纯度大大超过了类似的基于树脂的纯化。未来的工作目标是在聚合物载体中开发刷子改性膜，其孔径大小将允许渗透性的大幅增加。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载体兼容的新合成方法，也需要生长能够快速结合更多蛋白质的更厚的刷子。因此，蛋白质结合和非特异性吸附将作为刷子厚度、组成、密度和官能化以及膜组成和几何形状的函数来检查。有了新的膜，在大肠杆菌中表达的各种标记蛋白将得到纯化，包括SNAP-50、人MIP合成酶和SNAPc复合体。MIP合酶对肌醇的生物合成至关重要，而SNAP蛋白在基因表达中起关键作用。此外，SNAP-50提供了一个典型的亲和凝胶无法纯化的粘性蛋白质的例子。与公共卫生相关：这项研究将产生快速、廉价的方法来分离非常纯的蛋白质。这种技术将在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与健康相关的功能的研究中至关重要。

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.