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Production of Recombinant Salmon Calcitonin by In Vitro Amidation of an Escherichia coli Produced Precursor Peptide

Bio/Technology volume 11, pages 64–70 (1993)Cite this article

Abstract

Salmon calcitonin (sCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. We have produced salmon calcitonin by in vitro amidation of an E. coli produced precursor peptide. Glycine-extended sCT, the substrate for amidation, was produced in recombinant E. coli as part of a fusion with glutathione-S-transferase. The microbially produced soluble fusion protein was purified to near homogeneity by affinity chromatography. Following S-sulfonation of the fusion protein, the glycine-extended peptide was cleaved from the fusion by cyanogen bromide. The S-sulfonated peptide was recovered and enzymatically converted to the amidated peptide in a reaction with recombinant peptidylglycine α-amidating enzyme (α-AE) secreted from Chinese hamster ovary (CHO) cells. After reformation of the intramolecular disulfide bond, the sCT was purified with a step yield of 60%. The ease and speed of this recombinant process, as well as its potential for scale-up, make it adaptable to production demands for calcitonin, a proven useful agent for the treatment of post-menopausal osteoporosis. Moreover, the relaxed specificity of the recombinant α-AE for the penultimate amino acid which is amidated allows the basic process to be applied to the production of other amidated peptides.

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Author notes

  1. Arthur H. Bertelsent

    Present address: PharmaGenics, Inc., 4 Pearl Court, Allendale, NJ, 07401

  2. Martha V. L. Ray: Corresponding author.

Authors and Affiliations

  1. Molecular Biology Group, Unigene Laboratories, Inc., 110 Little Falls Road, Fairfield, NJ, 07004

    Martha V. L. Ray, Paul Van Duyne & Arthur H. Bertelsent

  2. Molecular Immunology Group, Unigene Laboratories, Inc., 110 Little Falls Road, Fairfield, NJ, 07004

    Dianne E. Jackson-Matthews & Amy M. Sturmer

  3. Analytical Chemistry Group, Unigene Laboratories, Inc., 110 Little Falls Road, Fairfield, NJ, 07004

    David J. Merkler, Angelo P. Consalvo & Stanley D. Young

  4. Protein Chemistry Group, Unigene Laboratories, Inc., 110 Little Falls Road, Fairfield, NJ, 07004

    James P. Gilligan & Paul P. Shields

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Ray, M., Duyne, P., Bertelsent, A. et al. Production of Recombinant Salmon Calcitonin by In Vitro Amidation of an Escherichia coli Produced Precursor Peptide. Nat Biotechnol 11, 64–70 (1993). https://doi.org/10.1038/nbt0193-64

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