Escherichia coli-derived human interferon-gamma with Cys-Tyr-Cys at the N-terminus is partially N alpha-acylated.
Purified preparations of recombinant human interferon-gamma (rIFN-gamma) with Cys-Tyr-Cys at the N-terminus ([ Cys-Tyr-Cys]IFN-gamma) 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-gamma without Cys-Tyr-Cys and rIFN-gamma 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-gamma is heterogeneous. To elucidate the structure of the N-terminal portion, the N-terminal peptide preparation was obtained by binding rIFN-gamma 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 alpha-formyl and N alpha-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-gamma is partially N alpha-acylated. The data also suggest that N alpha-acylation does not affect the biological activity of [Cys-Tyr-Cys]IFN-gamma.
Honda
S
S
Biotechnology Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Asano
T
T
Kajio
T
T
Nishimura
O
O
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins; D014443:Tyrosine
D000596:Amino Acids; D011994:Recombinant Proteins; D014443:Tyrosine; D007371:Interferon-gamma; D003545:Cysteine