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J Biol Chem

Title:		Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae
Author(s):		Gellerfors P; Axelsson K; Helander A; Johansson S; Kenne L; Lindqvist S; Pavlu B; Skottner A; Fryklund L;
Address:		"KABI, Stockholm, Sweden"
Journal Title:		J Biol Chem
Year:		1989
Volume:		264
Issue:		19
Page Number:		11444 - 11449
DOI:
ISSN/ISBN:		0021-9258 (Print) 0021-9258 (Linking)
Abstract:		"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 alpha 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 alpha-D-Manp(1----2)alpha-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"
Keywords:		"Actins/genetics Amino Acid Sequence Carbohydrate Sequence Chromatography, High Pressure Liquid Cloning, Molecular Electrophoresis, Polyacrylamide Gel Glycosylation Humans Insulin-Like Growth Factor I/genetics/*isolation & purification/metabolism Magnetic;"
Notes:		"MedlineGellerfors, P Axelsson, K Helander, A Johansson, S Kenne, L Lindqvist, S Pavlu, B Skottner, A Fryklund, L eng 1989/07/05 J Biol Chem. 1989 Jul 5; 264(19):11444-9"