RESUMO
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.
Assuntos
Calcitonina/biossíntese , Clonagem Molecular/métodos , Escherichia coli/genética , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Calcitonina/genética , Calcitonina/isolamento & purificação , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Cricetinae , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Glicina , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Salmão , TransfecçãoRESUMO
A sensitive competitive ELISA has been developed for the detection and quantitation of native and recombinant alpha-amidating enzyme. Chickens immunized with purified enzyme (75 kDa) isolated from a rat medullary thyroid carcinoma, produced IgY antibodies specific for the native enzyme. The assay is defined by a standard curve with a linear range of 0.78-12.5 ng/ml in phosphate-buffered saline, and a limit of sensitivity for detection of the enzyme of 0.20 ng/ml. The immunoassay is capable of detecting enzyme from both tumor derived sources, and from cells genetically engineered to secrete the enzyme into tissue culture medium containing up to 10% fetal calf serum.
