Separation and characterization of modified variants of recombinant human insulin-like growth factor I derived from a fusion protein secreted from Escherichia coli.
Human insulin-like growth factor I, IGF-I, was produced in Escherichia coli fused to a synthetic IgG-binding peptide The fusion protein is secreted into the medium during fermentation and was initially purified on an IgG-Sepharose column. After hydroxylamine cleavage, IGF-I was purified to homogeneity. During purification, impurities in the form of modified variants of IGF-I were detected and characterized. The closely related impurities were identified to be a misfolded form of IGF-I, having mismatched disulphide bonds, a form with the single methionine residue in IGF-I oxidized to methionine sulphoxide and a variant in which the methionine residue was substituted by a norleucine residue during protein synthesis. A form proteolytically cleaved between two arginine residue was also detected. These impurities were separated from the major component, native IGF-I, by using reverse-phase h.p.l.c. The modified molecules as well as native IGF-I were characterized both as intact molecules and as fragments, after pepsin digestion, using the techniques of plasma desorption m.s., N-terminal sequencing and amino acid analysis. The oxidized form was 90%, and the norleucine analogue was 70%, as potent as native IGF-I in a biological radioreceptor assay, and the form having mismatched disulphides lacked receptor affinity.
Forsberg
G
G
KabiGen AB, Stockholm, Sweden.
Palm
G
G
Ekebacke
A
A
Josephson
S
S
Hartmanis
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000032:analysis
D000596:Amino Acids; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D010434:Pepsin A