Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States.
J Am Chem Soc. 2012 Sep 26;134(38):15765-72. doi: 10.1021/ja303612d. Epub 2012 Sep 17.
A process for the preparation of an abiotic protein affinity ligand is described. The affinity ligand, a synthetic polymer hydrogel nanoparticle (NP), is formulated with functional groups complementary to the surface presentation of the target protein. An iterative process is used to improve affinity by optimizing the composition and proportion of functional monomers. Since the polymer NPs are formed by a kinetically driven process, the sequence of functional monomers in the polymer chain is not controlled; only the average composition can be adjusted by the stoichiometry of the monomers in the feed. To compensate for this the hydrogel NP is lightly cross-linked resulting in chain flexibility that takes place on a submillisecond time scale allowing the polymer to "map" onto a protein surface with complementary functionality. In this study, we report a lightly cross-linked (2%) N-isopropyl acrylamide (NIPAm) synthetic polymer NP (50-65 nm) incorporating hydrophobic and carboxylate groups that binds with high affinity to the Fc fragment of IgG. The affinity and amount of NP bound to IgG is pH dependent. The hydrogel NP inhibits protein A binding to the Fc domain at pH 5.5, but not at pH 7.3. A computational analysis was used to identify potential NP-protein interaction sites. Candidates include a NP binding domain that overlaps with the protein A-Fc binding domain at pH 5.5. The computational analysis supports the inhibition experimental results and is attributed to the difference in the charged state of histidine residues. Affinity of the NP (3.5-8.5 nM) to the Fc domain at pH 5.5 is comparable to protein A at pH 7. These results establish that engineered synthetic polymer NPs can be formulated with an intrinsic affinity to a specific domain of a large biomacromolecule.
一种非生物蛋白质亲和配体的制备方法。亲和配体是一种合成聚合物水凝胶纳米颗粒（NP），其功能基团与靶蛋白的表面呈现互补。通过优化功能单体的组成和比例，采用迭代过程来提高亲和力。由于聚合物 NPs 是由动力学驱动的过程形成的，因此聚合物链中的功能单体序列无法控制；只能通过进料单体的化学计量来调整聚合物的平均组成。为了弥补这一点，水凝胶 NP 被轻度交联，导致链的灵活性，这种灵活性发生在亚毫秒时间尺度上，使聚合物能够“映射”到具有互补功能的蛋白质表面。在这项研究中，我们报告了一种轻度交联（2%）的 N-异丙基丙烯酰胺（NIPAm）合成聚合物 NP（50-65nm），其含有疏水性和羧基基团，与 IgG 的 Fc 片段具有高亲和力。NP 与 IgG 的结合亲和力和结合量均依赖于 pH 值。在 pH 5.5 时，水凝胶 NP 抑制蛋白 A 与 Fc 结构域的结合，但在 pH 7.3 时则不抑制。计算分析用于鉴定潜在的 NP-蛋白相互作用位点。候选者包括一个 NP 结合域，该结合域在 pH 5.5 时与蛋白 A-Fc 结合域重叠。计算分析支持抑制实验结果，这归因于组氨酸残基的荷电状态不同。NP（3.5-8.5 nM）在 pH 5.5 时对 Fc 结构域的亲和力与 pH 7.3 时的蛋白 A 相当。这些结果表明，工程合成聚合物 NPs 可以被配方设计成与大生物大分子的特定结构域具有内在亲和力。
