Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae.
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.
Gellerfors
P
P
KABI, Stockholm, Sweden.
Axelsson
K
K
Helander
A
A
Johansson
S
S
Kenne
L
L
Lindqvist
S
S
Pavlu
B
B
Skottner
A
A
Fryklund
L
L
D000199:Actins / Q000235:genetics; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000032:analysis; D000072235:Mating Factor; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments; D010455:Peptides / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013002:Somatomedins / Q000302:isolation & purification*; D013912:Threonine / Q000378:metabolism; D014357:Trypsin
D000199:Actins; D010446:Peptide Fragments; D010455:Peptides; D021382:Protein Sorting Signals; D013002:Somatomedins; D013912:Threonine; D000072235:Mating Factor; D007334:Insulin-Like Growth Factor I; D014357:Trypsin; D008358:Mannose