Nagano Hayato, Mannen Teruhisa, Kikuchi Yoshimi, Shiraki Kentaro
Research Institute for Bioscience Product & Fine Chemicals, Ajinomoto Co, Inc., 1-1, Suzuki-cho, Kawasaki, 2108681, Japan; Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8573, Japan.
Research Institute for Bioscience Product & Fine Chemicals, Ajinomoto Co, Inc., 1-1, Suzuki-cho, Kawasaki, 2108681, Japan.
Protein Expr Purif. 2022 Aug;195-196:106091. doi: 10.1016/j.pep.2022.106091. Epub 2022 Mar 23.
Cell surface protein B (CspB) from Corynebacterium glutamicum has been developed as a reversible pH-responsive tag for protein purification. CspB fusion proteins precipitate at acidic pH, after that they completely dissolve at neutral pH. This property has been used in a non-chromatographic protein purification method named pH-responsive Precipitation-Redissolution of CspB tag Purification (pPRCP). However, it is difficult to apply pPRCP to proteins that are unstable under acidic conditions. In an effort to shift the precipitation pH to a milder range, we investigated the solution conditions of CspB-fused Teriparatide (CspB50TEV-Teriparatide) during the process of pH-responsive precipitation using pPRCP. The purified CspB50TEV-Teriparatide in buffer without additives precipitated at pH 5.3. By contrast, CspB50TEV-Teriparatide in buffer with 0.5 M NaSO precipitated at pH 6.6 because of the kosmotropic effect. Interestingly, the pH at which precipitation occurred was independent of the protein concentration. The precipitated CspB50TEV-Teriparatide was fully redissolved at above pH 8.0 in the presence or absence of salt. The discovery that proteins can be precipitated at a mild pH will allow pPRCP to be applied to acid-sensitive proteins.
谷氨酸棒杆菌的细胞表面蛋白B（CspB）已被开发为一种用于蛋白质纯化的可逆pH响应标签。CspB融合蛋白在酸性pH下沉淀，之后在中性pH下完全溶解。这一特性已被用于一种名为CspB标签纯化的pH响应沉淀-再溶解（pPRCP）的非色谱蛋白质纯化方法中。然而，将pPRCP应用于在酸性条件下不稳定的蛋白质是困难的。为了将沉淀pH转移到更温和的范围，我们在使用pPRCP进行pH响应沉淀的过程中研究了CspB融合的特立帕肽（CspB50TEV-特立帕肽）的溶液条件。在没有添加剂的缓冲液中纯化的CspB50TEV-特立帕肽在pH 5.3时沉淀。相比之下，由于向心作用，在含有0.5 M NaSO的缓冲液中的CspB50TEV-特立帕肽在pH 6.6时沉淀。有趣的是，发生沉淀的pH与蛋白质浓度无关。沉淀的CspB50TEV-特立帕肽在有盐或无盐的情况下在pH 8.0以上完全重新溶解。蛋白质可以在温和的pH下沉淀这一发现将使pPRCP能够应用于对酸敏感的蛋白质。
