Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli.
Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.
Wadensten
H
H
KabiGen AB, S-112 87 Stockholm, Sweden.
Ekebacke
A
A
Hammarberg
B
B
Holmgren
E
E
Kalderén
C
C
Tally
M
M
Moks
T
T
Uhlén
M
M
Josephson
S
S
Hartmanis
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D013058:Mass Spectrometry; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012997:Solvents
D000596:Amino Acids; D011993:Recombinant Fusion Proteins; D012997:Solvents; D007335:Insulin-Like Growth Factor II