Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States.
Tsukuba Research Laboratories, JSR Corporation, Ibaraki 305-0841, Japan.
Biomacromolecules. 2021 Jun 14;22(6):2641-2648. doi: 10.1021/acs.biomac.1c00335. Epub 2021 May 19.
We describe an approach for the discovery of protein affinity reagents (PARs). Abiotic synthetic hydrogel copolymers can be "tuned" for selective protein capture by the type and ratios of functional monomers included in their polymerization and by the polymerization conditions (i.e., pH). By screening libraries of hydrogel nanoparticles (NPs) containing charged and hydrophobic groups against a protein target (IgG), a stimuli-responsive PAR is selected. The robust carbon backbone synthetic copolymer is rapidly synthesized in the chemistry laboratory from readily available monomers. The production of the PAR does not require living cells and is free from biological contamination. The capture and release of the protein by the copolymer NP is reversible. IgG is sequestered from human serum at pH 6.5 and following a wash step, the purified protein is released by elevating the pH to 7.3. The binding and release of the protein occur without denaturation. The abiotic material functions as a selective PAR for the F(ab') domain of IgG for pull-down and immunoprecipitation experiments and for isolation and purification of proteins from complex biological mixtures.
我们描述了一种用于发现蛋白质亲和试剂 (PARs) 的方法。通过在聚合过程中包含的功能单体的类型和比例以及聚合条件（即 pH 值），可以对非生物合成水凝胶共聚物进行“调谐”，以实现对蛋白质的选择性捕获。通过针对蛋白质靶标（IgG）筛选含有带电和疏水基团的水凝胶纳米颗粒 (NP) 文库，选择了一种对刺激有响应的 PAR。这种坚固的碳骨架合成共聚物可以在化学实验室中通过易得的单体快速合成。PAR 的生产不需要活细胞，也不会受到生物污染。该共聚物 NP 对蛋白质的捕获和释放是可逆的。在 pH 值为 6.5 时从人血清中分离 IgG，然后通过将 pH 值升高到 7.3 进行洗涤步骤，即可释放纯化的蛋白质。蛋白质的结合和释放不会导致变性。这种非生物材料可以作为 IgG 的 F(ab') 结构域的选择性 PAR，用于下拉和免疫沉淀实验，以及从复杂的生物混合物中分离和纯化蛋白质。
