Author： Merli S. de Falco S. Verdoliva A. Tortora M. Villain M. Silvi P. Cassani G. Fassina G.
Publisher： Academic Press
ISSN： 1046-5928
Source： Protein Expression and Purification, Vol.7, Iss.4, 1996-06, pp. : 347-354
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Abstract
Amidating mouse pituitary cells (AtT-20) have been engineered to secrete human calcitonin (hCT) in the fully active amidated form, without the need of additional enzymatic or chemical modifications. The 141-residue human calcitonin precursor has first been cloned in the eucaryotic expression vector pRc/RSV, and the resulting plasmid pRc/RSV/hCT introduced in AtT-20 cells. After transfection, 122 independent clones resistant to G-418 were selected and screened for calcitonin production using a competitive ELISA specifically designed to detect the amidated form of calcitonin. One of these clones was amplified and showed expression of 17 ng/ml of hCT, with a 70% increase in productivity after cAMP treatment. Calcitonin was partially purified from culture medium by two sequential steps of reverse-phase chromatography and characterized in terms of immunoreactivity and molecular weight by TOF-MALDI mass spectroscopy, which confirmed the intended chemical nature and the presence of the C-terminal amidated residue.
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