Production and characterization of recombinant chicken insulin-like growth factor-I from Escherichia coli.
Recombinant chicken insulin-like growth factor-I (cIGF-I) has been produced in Escherichia coli after first modifying a plasmid that coded for a human IGF-I (hIGF-I) fusion protein, in order to introduce codons for the eight amino acid substitutions. The cIGF-I fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography to remove proteinases, refolded and partially purified by reverse-phase high-performance liquid chromatography. The fusion protein was cleaved with hydroxylamine after which cIGF-I was purified to homogeneity by three additional chromatographic steps. Recombinant cIGF-I was equipotent with hIGF-I in cell culture bioassays of protein synthesis and breakdown using rat L6 myoblasts and chick embryo fibroblasts. Binding of radiolabelled cIGF-I and hIGF-I was also equivalent in the two cell lines, as was their binding in ligand blots of chicken, sheep and human plasma. The cross-reactivity of cIGF-I in a polyclonal hIGF-I radioimmunoassay was 60% of that observed with hIGF-I. The availability of recombinant cIGF-I will facilitate investigations into the role of IGF-I in chicken growth and development.
Upton
F Z
FZ
Cooperative Research Centre for Tissue Growth and Repair, CSIRO Division of Human Nutrition, Adelaide, South Australia.
Francis
G L
GL
Ross
M
M
Wallace
J C
JC
Ballard
F J
FJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I