Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans.
Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat alpha-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.
D000818:Animals; D001681:Biological Assay; D002116:Calcitonin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013302:Streptomyces / Q000235:genetics*
D011994:Recombinant Proteins; C028815:salmon calcitonin; D002116:Calcitonin