Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide.
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 alpha-amidating enzyme (alpha-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 alpha-AE for the penultimate amino acid which is amidated allows the basic process to be applied to the production of other amidated peptides.
Ray
M V
MV
Unigene Laboratories, Inc., Fairfield, NJ 07004.
Van Duyne
P
P
Bertelsen
A H
AH
Jackson-Matthews
D E
DE
Sturmer
A M
AM
Merkler
D J
DJ
Consalvo
A P
AP
Young
S D
SD
Gilligan
J P
JP
Shields
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002116:Calcitonin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D005998:Glycine; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D012474:Salmon; D014162:Transfection
D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002116:Calcitonin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005982:Glutathione Transferase; D005998:Glycine