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.