High expression of a recombinant human calcitonin precursor peptide in Escherichia coli.
Human calcitonin (hCT) is a C-terminus alpha-amidated peptide hormone consisting of 32 amino acids. The amidated structure is essential for its biological activities, and the C-terminal-glycine-extended precursor peptide, hCT[G], is converted to bioactive hCT by a C-terminus-alpha-amidating enzyme. An efficient production method is described for the hCT[G] peptide, as a part of the fusion protein consisting of a modified E. coli beta-galactosidase, linker amino acids and hCT[G]. Stable inclusion bodies of the fusion protein in E. coli were expressed by focusing on the amino acid charge, and the fusion protein was modified by inserting a basic amino acid sequence into its linker region. This modification greatly affected the formation of inclusion bodies. E. coli strain W3110/pG97S4DhCT [G]R4 could produce a large amount of stable inclusion bodies, and the hCT[G] peptide was released quantitatively from the fusion protein by S. aureus V8 protease. This enabled a large-scale production method to be established for the hCT[G] precursor peptide in E. coli to produce mature hCT.
Yabuta
M
M
Suntory Bio-Pharma Tech Center, Gunma, Japan.
Suzuki
Y
Y
Ohsuye
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D002116:Calcitonin / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D002116:Calcitonin; D004247:DNA