The pH-responsive precipitation-redissolution of the CspB fusion protein, CspB50TEV-Teriparatide, triggered by changes in secondary structure.

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 ß-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 ß-sheet structure in response to decreasing pH, and the growth of the precipitate particles occurred through intermolecular interactions.

Solution design to extend the pH range of the pH-responsive precipitation of a CspB fusion protein.

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 Na2SO4 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.

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012.

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.