Production of a human calcitonin precursor with Staphylococcus carnosus: Secretory expression and single-step recovery by expanded bed adsorption

Recombinant human calcitonin (hCT) can be expressed by Staphylococcus carnosus as a precursor peptide being part of a secreted fusion protein which contains the propeptide of a Staphylococcus hyicus lipase as secretion carrier and multiple repeats of the hCT precursor. Fusion proteins containing two (PhCT2), four (PhCT4), or eight (PhCT8) hCT precursors were expressed. Due to the unfavourable biochemical properties of PhCT4 and PhCT8, PhCT2 was the most efficient fusion protein for a production process. Expression from a xylose-inducible promoter from Staphylococcus xylosus resulted in a maximum secretory production of PhCT2, since the amount of PhCT2 synthesized in total corresponded almost precisely to the upper limit of the capacity of the cellular protein secretion apparatus. An expanded bed adsorption procedure for the recovery of PhCT2 from unclarified culture broth was established using the cation exchanger STREAMLINE SP at pH 5. This integrated operation allows a single-step recovery of PhCT2 and was successfully transferred to a pilot scale (100 1 culture broth).