Abstract
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).
Highlights
Recombinant human parathyroid hormone(l-84) was obtained from Escherichia coli using a cleavable fusion protein strategy
Calcium ion homeostasis in vertebrates is regulated primarily through the action of parathyroid hormone (PTH).l In humans, PTH is synthesized as a 1%amino acid precursor polypeptide, which is processed by the endoplasmic reticulum/ Golgi apparatus and secreted as an 84-amino acid peptide (Potts et al, 1980)
We have described a high yield system for producing hPTH
Summary
Pkxsmid Construction-The plasmid construction scheme is outlined in Fig. 1. The hGH expression vector pGH-L9 (Ikehara et al., 1984), kindly provided by Dr Tomoko Doi (Massachusetts Institute of Technology), contains the hGH cDNA downstream from the E. Procedures.” Fiss. 4 and 5. and Table I) are uresentedin miniurint at the end of this paper. Miniprint is easily read with the aid o$ a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. This is an Open Access article under the CC BY license.
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