Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein

• Published in: The Journal of biological chemistry 1990-09, Vol.265 (26), p.15854-15859
• Main Authors: T J Gardella, D Rubin, A B Abou-Samra, H T Keutmann, J T Potts, Jr, H M Kronenberg, S R Nussbaum
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Biotechnology; Cell Line; Chromatography, High Pressure Liquid; coagulation factor X; Cyclic AMP - metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Factor X - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic engineering; Genetic technics; Humans; Methods. Procedures. Technologies; Molecular Sequence Data; Molecular Weight; Osteosarcoma; Parathyroid Hormone - genetics; Parathyroid Hormone - isolation & purification; Parathyroid Hormone - pharmacology; Plasmids; Rats; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - pharmacology; Restriction Mapping; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)55477-6

Recombinant human parathyroid hormone (hPTH)-(1-84) was obtained from Escherichia coli using a cleavable fusion protein strategy. The fusion protein contains residues 1-138 of human growth hormone as the amino-terminal region and residues 1-84 of hPTH as the carboxyl-terminal region. A 7-residue linker containing the recognition/cleavage sequence of the site-specific blood coagulation protease activated factor X (factor Xa) joins the two regions. Intact hPTH-(1-84) is released from this fusion protein by cleavage in vitro with factor Xa. The fusion protein was produced at a high level and formed inclusion bodies which allowed it to be easily purified by low speed centrifugation, with a yield of approximately 50 mg/liter of culture. After factor Xa cleavage and high performance liquid chromatography purification, highly purified hPTH was obtained, with a final yield of 1.5-3 mg/liter. Physical and biological characterization of the purified hormone demonstrated that it was intact and active hPTH-(1-84).