Recombinant chicken IFN-gamma expressed in Escherichia coli: analysis of C-terminal truncation and effect on biologic activity.
Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties. Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN. Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli. Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively). Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence. Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma. Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133. It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141. A truncated recombinant construct (His1-Lys133) was also expressed in E. coli and had biologic activity comparable with that of the full-length construct. The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures. Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32%.
Michalski
W P
WP
CSIRO, Animal Health, Australian Animal Health Laboratory, Geelong, Victoria. Wojtek.Michalski@dah.csiro.aw
Shiell
B J
BJ
O'Neil
T E
TE
Beddome
G
G
Lowenthal
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D011994:Recombinant Proteins; D007371:Interferon-gamma