Cell surface protein B (CspB) from Corynebacterium glutamicum is used as a pH-responsive peptide tag to enable a simple solid-liquid separation method for isolating a CspB fusion protein. Here we demonstrate the first application of a CspB tag for the purification of Teriparatide， which is a biologic drug that is prescribed for osteoporosis. The Teriparatide was constructed as CspB50TEV-Teriparatide， comprising 50 amino acid residues of CspB， the cleavage site of TEV protease， and Teriparatide. CspB50TEV-Teriparatide was expressed in a culture supernatant by C. glutamicum secretion system at 3.0 g/L (equivalent to approximately 1.2 g/L Teriparatide). The CspB50TEV-Teriparatide was precipitated by re... More
Cell surface protein B (CspB) from Corynebacterium glutamicum is used as a pH-responsive peptide tag to enable a simple solid-liquid separation method for isolating a CspB fusion protein. Here we demonstrate the first application of a CspB tag for the purification of Teriparatide， which is a biologic drug that is prescribed for osteoporosis. The Teriparatide was constructed as CspB50TEV-Teriparatide， comprising 50 amino acid residues of CspB， the cleavage site of TEV protease， and Teriparatide. CspB50TEV-Teriparatide was expressed in a culture supernatant by C. glutamicum secretion system at 3.0 g/L (equivalent to approximately 1.2 g/L Teriparatide). The CspB50TEV-Teriparatide was precipitated by reducing the pH of the culture supernatant， and the precipitate was then dissolved in a neutral buffer. A TEV protease treatment was applied to cleave the Teriparatide from the CspB50TEV-Teriparatide. Then， the remaining digested CspB50TEV， undigested CspB50TEV-Teriparatide， and TEV protease were precipitated in an acidic pH， whereas the soluble Teriparatide remained in the supernatant. The process had a yield of 96.5% and resulted in Teriparatide with a purity of 98.0% and productivity of 1.1 g/L of C. glutamicum culture. Thus， tag-free Teriparatide was successfully purified from the CspB fusion protein using only pH changes， centrifugation， and protease digestion without the need for chromatography. This versatile purification protocol is expected to be applicable to various proteins from laboratory to industrial scales.
