A NOVEL-APPROACH FOR HIGH-LEVEL PRODUCTION OF A RECOMBINANT HUMAN PARATHYROID-HORMONE FRAGMENT IN ESCHERICHIA-COLI

We describe a novel approach to the production in E. coli of a peptide fragment derived from the human parathyroid hormone (hPTH). The first 38 amino acids of hPTH were fused at the amino terminus to a derivati ve of the bacteriophage T4-encoded gp55 protein, and were expressed in the E. coli cytoplasm in inclusion bodies at levels exceeding 50% of the total cell protein. Solubilization and subsequent incubation of th e inclusion bodies in dilute hydrochloric acid facilitated the cleavag e of an acid-labile aspartyl-prolyl peptide bond engineered into the f usion protein, thus releasing the hormone fragment directly from the i nclusion body preparation. The amino-terminal prolyl-prolyl dipeptide- extension was subsequently removed by treatment with Lactococcus lacti s dipeptidyl peptidase IV which was overexpressed in E. coli and purif ied to near homogeneity from the cytosol of the recombinant bacteria. In pilot-scale fermentations, more than 80 mg of pure hPTH(1-38) were isolated per liter of bacterial culture, with an overall yield of 35%. This process is suitable for scale-up, is cost effective, and by empl oying recombinant dipeptidyl peptidase IV, should be widely and direct ly applicable to the manufacturing of peptides of pharmaceutical inter est.