Reduction studies on bacterial recombinant somatomedin C/insulin-like growth factor-1.
N-Met-Somatomedin C/insulin-like growth factor-1 (rSmC) had been produced in recombinant Escherichia coli in monomeric form. The intact rSmC peptide is initially synthesized in E. coli cells in denatured form as inclusion bodies. The rSmC peptide in these inclusion bodies was found in reduced form. Isolation of this rSmC peptide was accomplished by separation and dissolution of the inclusion bodies, with dissociation of non-covalently aggregated species. The reduced rSmC was converted to a metastable state, termed un-refolded rSmC. Further processing of this rSmC generated two other isomers, termed refolded rSmC. The transitions of the peptide among these different states, reduced rSmC, un-refolded rSmC, and refolded rSmC can be readily monitored by reversed-phase high-performance liquid chromatography. By reduction and re-oxidation of the purified individual isomers we found that they are likely to be related to each other as conformation isomers which appear to be stabilized by disulfide bonds.
Meng
H
H
Northbrook Laboratories, Pitman-Moore Inc., IL 60062.
Burleigh
B D
BD
Kelly
G M
GM
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis*; D007525:Isoelectric Focusing; D007536:Isomerism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000032:analysis; D013002:Somatomedins / Q000032:analysis*
D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I