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.
Biochem Biophys Rep. 2023 Jan 26;33:101435. doi: 10.1016/j.bbrep.2023.101435. eCollection 2023 Mar.
Cell surface protein B (CspB) fusion proteins can undergo reversible pH-responsive precipitation-redissolution. A pH-responsive precipitation-redissolution of CspB tag purification (pPRCP) method was established for protein purification using this property. However, the mechanism of the pH-responsive precipitation of CspB fusion proteins is unknown, which has made it difficult to set process parameters for pPRCP. In this study, we investigated the mechanism of the pH-responsive precipitation of CspB fusion proteins using CspB50TEV-Teriparatide (CspB-teri) as a model. As expected, CspB-Teri was reversibly precipitated at acidic pH. By contrast, CspB-Teri was not precipitated under unfolding conditions induced by trifluoroethanol, urea, or guanidine hydrochloride, even at acidic pH. The conformation of CspB-Teri changed to a β-sheet-rich structure as the pH decreased, followed by the formation of intermolecular interactions, which caused precipitation. The particle size of the CspB-Teri precipitate increased in a protein concentration-dependent manner. These results indicated that the pH-responsive precipitation of CspB-Teri is triggered by the formation of a β-sheet structure in response to decreasing pH, and the growth of the precipitate particles occurred through intermolecular interactions.
细胞表面蛋白B（CspB）融合蛋白可发生可逆的pH响应沉淀-再溶解。利用这一特性建立了一种用于蛋白质纯化的CspB标签纯化（pPRCP）方法，即pH响应沉淀-再溶解法。然而，CspB融合蛋白pH响应沉淀的机制尚不清楚，这使得难以设定pPRCP的工艺参数。在本研究中，我们以CspB50TEV-特立帕肽（CspB-特立帕肽）为模型，研究了CspB融合蛋白pH响应沉淀的机制。正如预期的那样，CspB-特立帕肽在酸性pH下可逆沉淀。相比之下，即使在酸性pH下，CspB-特立帕肽在三氟乙醇、尿素或盐酸胍诱导的变性条件下也不会沉淀。随着pH值降低，CspB-特立帕肽的构象转变为富含β-折叠的结构，随后形成分子间相互作用，导致沉淀。CspB-特立帕肽沉淀物的粒径以蛋白质浓度依赖性方式增加。这些结果表明，CspB-特立帕肽的pH响应沉淀是由pH值降低时β-折叠结构的形成触发的，沉淀颗粒的生长是通过分子间相互作用发生的。
