High expression of a recombinant human calcitonin precursor peptide in Escherichia coli |
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Authors: | M. Yabuta Y. Suzuki K. Ohsuye |
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Affiliation: | (1) Suntory Bio-Pharma Tech Center, 2716-1 Kurakake, Akaiwa, Chiyoda-machi, 370-05 Ohra-Gun, Gunma, Japan |
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Abstract: | Human calcitonin (hCT) is a C-terminus -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- -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 -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. |
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