| Autor: |
Ray, Martha V. L., Duyne, Paul Van, Bertelsent, Arthur H., Jackson-Matthews, Dianne E., Sturmer, Amy M., Merkler, David J., Consalvo, Angelo P., Young, Stanley D., Gilligan, James P., Shields, Paul P. |
| Zdroj: |
Bio/Technology; January 1993, Vol. 11 Issue: 1 p64-70, 7p |
| Abstrakt: |
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. |
| Databáze: |
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