Gellerfors P, Axelsson K, Helander A, Johansson S, Kenne L, Lindqvist S, Pavlu B, Skottner A, Fryklund L Isolation and characterization of a glycosylated form of human insulin like growth factor I produced in Saccharomyces cerevisiae Journal of Biological Chemistry264 (1989)
11444-11449
NCBI PubMed ID:2500441 Journal NLM ID:2985121R Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology Institutions: KABI, Stockholm, Sweden
Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF α 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be α-D-Manp(1----2)α-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.
Methods: 13C NMR, 1H NMR Comments, role: Parent molecule: human insulin like growth factor I;
Related record ID(s): 124753 NCBI Taxonomy refs (TaxIDs):4932 Reference(s) to other database(s): CCSD:42661, CBank-STR:4619, OGlycBase:103, SwissProt:P00695 Show glycosyltransferases
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