Escherichia coli-derived human interferon-γ with CysTyrCys at the N-terminus is partially N α-acylated

Abstract

Purified preparations of recombinant human interferon-γ (rIFN-γ) with CysTyrCys at the N-terminus ([CysTyrCys]IFN-γ) derived from Escherichia coli gave two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two peaks on reversed-phase high-performance liquid chromatography (rpHPLC). In contrast, rIFN-γ without CysTyrCys and rIFN-γ in which both Cys-1 and Cys-3 were substituted with serine behaved as a single species on both SDS-PAGE and rpHPLC. These results suggest that the N-terminal portion of rIFN-γ is heterogeneous. To elucidate the structure of the N-terminal portion, the N-terminal peptide preparation was obtained by binding rIFN-γ to thiopropyl-Sepharose 6B gel with disulfide linkage followed by trypsin digestion and elution with 2-mercaptoethanol. The preparation gave four peaks (NT-1, NT-2, NT-3, and NT-4, in order of elution) on rpHPLC; all four were found to be Cys-1-Lys-9 by amino acid analysis after acid hydrolysis. Various analyses indicate that NT-1 is the intact nonapeptide, that NT-3 and NT-4 are N α-formyl and N α-acetyl forms of NT-1, respectively, and that NT-2 may be S-blocked at Cys-1. It is concluded that E. coli-derived [CysTyrCys]IFN-γ is partially N α-acylated. The data also suggest that N α-acylation does not affect the biological activity of [CysTyrCys]-IFN-γ.