Department of Chemistry, University of California, Irvine, California 92697, United States.
Department of Chemistry, Missouri State University, 901 South National Avenue, Springfield, Missouri 65897, United States.
Biomacromolecules. 2021 Apr 12;22(4):1695-1705. doi: 10.1021/acs.biomac.1c00119. Epub 2021 Mar 30.
We report a metal free synthetic hydrogel copolymer with affinity and selectivity for His6-tagged peptides and proteins. Small libraries of copolymers incorporating charged and hydrophobic functional groups were screened by an iterative process for His6 peptide affinity. The monomer selection was guided by interactions found in the crystal structure of an anti-His tag antibody-His6 peptide antigen complex. Synthetic copolymers incorporating a phenylalanine-derived monomer were found to exhibit strong affinity for both His6-containing peptides and proteins. The proximity of both aromatic and negatively charged functional groups were important factors for the His6 affinity of hydrogel copolymers. His6 affinity was not compromised by the presence of enzyme cleavage sequences. The His6-copolymer interactions are pH sensitive: the copolymer selectively captured His6 peptides at pH 7.8 while the interactions were substantially weakened at pH 8.6. This provided mild conditions for releasing His6-tagged proteins from the copolymer. Finally, a synthetic copolymer coated chromatographic medium was prepared and applied to the purification of a His6-tagged protein from an expression system. The results establish that a synthetic copolymer-based affinity medium can function as an effective alternative to immobilized metal ion columns for the purification of His6-tagged proteins.
我们报道了一种无金属的合成水凝胶共聚物，它对 His6 标记的肽和蛋白质具有亲和力和选择性。通过迭代过程筛选了带有电荷和疏水性官能团的共聚小文库，以获得 His6 肽亲和力。单体选择是根据抗 His 标签抗体- His6 肽抗原复合物的晶体结构中发现的相互作用来指导的。含有苯丙氨酸衍生单体的合成共聚物被发现对含 His6 的肽和蛋白质都具有很强的亲和力。芳香族和带负电荷官能团的接近性是水凝胶共聚物 His6 亲和力的重要因素。酶切序列的存在并不影响 His6 亲和力。His6-共聚物相互作用对 pH 敏感：共聚物在 pH 7.8 时选择性地捕获 His6 肽，而在 pH 8.6 时相互作用大大减弱。这为从共聚物中释放 His6 标记的蛋白质提供了温和的条件。最后，制备了一种合成共聚体涂覆的色谱介质，并将其应用于从表达系统中纯化 His6 标记的蛋白质。结果表明，基于合成共聚物的亲和介质可以作为固定化金属离子柱的有效替代品，用于纯化 His6 标记的蛋白质。
