Characterization of E. coli-Derived Recombinant Human Interferon-β as Compared with Fibroblast Human Interferon-β

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Abstract
Homogeneous E. coli -derived recombinant human interferon-β ( E. coli -rHulFN-β) was characterized in order to elucidate its physicochemical properties, as compared with those of fibroblast human interferon-β (fibroblast HulFN-β). Purified E. coli -rHulFN-β and fibroblast HuIFN-β exhibited a single band of M r 19,000 and 23,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The primary structure of E. coli-rHuIFN-β was identical to the prediction from the cDNA sequence. Furthermore, both the circular dichroism (CD) spectra and the 1 H nuclear magnetic resonance (NMR) spectra of E. coli -rHuIFN-β and fibroblast HuIFN-β at pH 6.8 were closely similar to each other. On the other hand, on reverse-phase high-performance liquid chromatography (HPLC) using a C18 column, the retention time of E. coli -rHuIFN-β was longer than that of fibroblast HuIFN-β. Moreover, although the isoelectric point of E. coli -rHuIFN-β was pH 8.9, purified fibroblast HuIFN-β exhibited multiple isoelectric points, probably due to heterogeneity of the carbohydrate moiety. These results indicate that the E. coli -rHuIFN-β polypeptide folds similarly to fibroblast HuIFN-β, and the carbohydrate moiety of natural HuIFN-β has little influence on higher-order structure but does influence the hydrophobic and the electrostatic properties of the molecule.