PDF(Pubmed)
Abstract：
While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.
摘要：
镍-次氮基三乙酸(Ni-NTA)在重组蛋白纯化方面有很大的进步,其局限性,包括某些蛋白质的非特异性结合和部分纯化，强调额外的纯化，如尺寸排阻和离子交换色谱的必要性。然而,通常需要诸如FPLC的专用设备，但在许多实验室中并不常见。这里,我们展示了一种利用多磷酸盐（polyP）通过非共价相互作用纯化具有组氨酸重复的蛋白质的新方法。我们的研究表明，固定化的polyP在5.5-7.5的pH范围内有效地结合组氨酸标记的蛋白质，即使在还原剂DTT和螯合剂EDTA存在下也能保持结合效力。我们进行了从Ni-NTA后的细胞裂解物和级分中纯化各种蛋白质的实验。我们的结果表明，polyP树脂能够在Ni-NTA后进一步纯化，而无需专用设备，也不会损害蛋白质活性。这种成本有效且方便的方法提供了一种可行的方法，作为Ni-NTA的补充方法。
