Semisynthesis of human ghrelin: condensation of a Boc-protected recombinant peptide with a synthetic O-acylated fragment.
The creation of peptide using a combination of recombinant expression and chemical synthesis can be a powerful tool for the production of a wide variety of polypeptides modified by phosphorylation, glycosylation, etc. We have developed a new method for the preparation of a recombinant peptide with a free N(alpha)-amino group and protected N(epsilon)-amino groups, and have used this method in the semisynthesis of human ghrelin. Ghrelin, a natural ligand for growth hormone secretagogue receptor, is a 28-residue peptide with an essential n-octanoyl modification on Ser3. A 7-residue N-terminal fragment of ghrelin containing the octanoyl modification was prepared by Fmoc chemistry. In the preparation of it, all reactions were performed on the 2-chlorotrityl resin. Additionally, TBDMS and tBu turned out to be the most effective protection groups for the Ser3 and the Ser2, Ser6, respectively. For preparation of a 21-residue C-terminal fragment, we established a two-step protease processing method for the partially protected segment. A recombinant precursor peptide was Boc protected and subsequently cleaved using two distinct proteases, OmpT and Kex2. The peptides were then coupled to each other and, after deprotection, resulted in fully active human ghrelin.
Makino
Tomohiro
T
Institute for Medicinal Research and Development, Daiichi Suntory Pharma Co., Ltd., Ohra, Gunma 370-0503, Japan. tomohiro_makino@asubio.co.jp [corrected]
Matsumoto
Masaru
M
Suzuki
Yuji
Y
Kitajima
Yasuo
Y
Yamamoto
Katsuhiko
K
Kuramoto
Masashi
M
Minamitake
Yoshiharu
Y
Kangawa
Kenji
K
Yabuta
Masayuki
M
D000215:Acylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D002210:Caprylates / Q000737:chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D054439:Ghrelin; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017646:Organosilicon Compounds / Q000737:chemistry; D036361:Peptide Hormones / Q000096:biosynthesis* / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D001704:Biopolymers; D002210:Caprylates; D054439:Ghrelin; D008024:Ligands; D017646:Organosilicon Compounds; D036361:Peptide Hormones; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C009660:t-butyldimethylsilyl compounds; D012694:Serine; D001616:beta-Galactosidase; D008239:Lysine; C031492:octanoic acid; D002118:Calcium