A simple and efficient scheme for the purification of insulin-like growth factor II from human bone matrix extract.
Human insulin-like growth factor II (IGF-II) has been purified to homogeneity from bone which contained 10-15 times more IGF-II than insulin-like growth factor-I (IGF-I). After extraction of IGF-II by demineralization of human bone powder with 10% EDTA containing 4M guanidine HCl at pH 7.4, IGF-II was separated from IGF binding proteins by hydroxylapatite chromatography in the presence of 4M guanidine HCl. The hydroxylapatite unbound fraction containing IGF-II was purified by affinity chromatography using Sm 1.2. monoclonal antibodies, which bind both IGF-I and IGF-II. The final purification of IGF-II was achieved by FPLC mono S ion-exchange chromatography in which IGF-II was separated from IGF-I. Human IGF-II thus purified was shown to be pure by (1) HPLC reverse-phase chromatography, (2) SDS-PAGE, and (3) N-terminal amino acid sequence. From 300 g of bone, 0.18 mg IGF-II was obtained with an overall recovery of 42%. These studies demonstrate the usefulness of (1) bone as a source of IGF-II purification and (2) antibodies that cross-react with both IGF-I and IGF-II for affinity purification of IGFs.
Mohan
S
S
Department of Medicine, Loma Linda University, California.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001857:Bone Matrix / Q000032:analysis*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000302:isolation & purification; D007335:Insulin-Like Growth Factor II / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D013002:Somatomedins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II