Structure and activity dependence of recombinant human insulin-like growth factor II on disulfide bond pairing.
The complete peptide map of purified folded recombinant human insulin-like growth factor II (rhIGF-II) was determined to verify its sequence and disulfide bonding scheme. Each peptide generated by digestion with pepsin was purified and characterized by amino acid analysis, amino acid sequence analysis, and fast atom bombardment/mass spectrometry. Some peptides were also sequenced using tandem mass spectrometry. The rhIGF-II peptide map was compared to that of rat insulin-like growth factor II and to that of a disulfide-bonded isomer of rhIGF-II. The data obtained in these studies are consistent with the conclusion that the rhIGF-II obtained from Escherichia coli has the correct amino acid composition, sequence, and the native disulfide-bonded structure. The binding affinities of these forms of recombinant IGF-II for IGF carrier proteins were measured in an IGF binding protein assay. The disulfide isomer of rhIGF-II was 160-fold less potent than native rhIGF-II in the competitive protein binding assay. These studies illustrate the need to characterize recombinant polypeptides containing disulfide bonds to allow the native structure to be verified before characterizing the biological properties of such molecules in hopes of elucidating their physiologic functions.
Smith
M C
MC
Department of Biochemistry, Eli Lilly and Company, Indianapolis, Indiana 46285.
Cook
J A
JA
Furman
T C
TC
Occolowitz
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000302:isolation & purification* / Q000378:metabolism; D007536:Isomerism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013002:Somatomedins / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D014357:Trypsin
D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II; D014357:Trypsin