An expression system for the single-step production of recombinant human amidated calcitonin.
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.
Merli
S
S
Protein Engineering Unit, Tecnogen S.C.p.A., Parco Scientifico, Piana di Monte Verna (CE), Italy.
De Falco
S
S
Verdoliva
A
A
Tortora
M
M
Villain
M
M
Silvi
P
P
Cassani
G
G
Fassina
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001483:Base Sequence; D003994:Bucladesine / Q000378:metabolism; D002116:Calcitonin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004274:DNA, Recombinant / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014162:Transfection
D017931:DNA Primers; D004274:DNA, Recombinant; D007106:Immune Sera; D011994:Recombinant Proteins; D003994:Bucladesine; D002116:Calcitonin; D001205:Ascorbic Acid