Structure-function analysis of the human interferon gamma. The COOH terminus is not essential for functional activity.
The structure-function relationships for the human interferon gamma (HuIFN-gamma) were studied using recombinant variants that had various deletions at the carboxyl terminus. Four COOH-terminal deletion variants were constructed that contained the amino-terminal 122, 117, 111, and 106 amino acid residues. These variants were constructed by specific DNA modifications and were expressed in Escherichia coli. The deletion of 21 amino acid residues resulted in only 2- and 3-fold reduction in the antiviral and antiproliferative specific activities, respectively. Thus, the carboxyl-terminal 21 residues are not directly involved in the function of the HuIFN-gamma. The level of intracellular accumulation was also decreased by 3-5-fold. Further deletions of 26, 32, and 37 residues from the COOH terminus resulted in the lack of detectable activity as well as in 50-100-fold reduction in the level of accumulation in the bacterial cell. However, each of the modified plasmids was found to have comparable efficiency in directing the production of the respective variant molecules relative to the full-length HuIFN-gamma molecule in an in vitro transcription-translation assay. Thus, the failure of some of the deletion variant molecules to accumulate in the E. coli cell is likely due to their instability in vitro. The loss of the COOH-terminal 21 amino acid residues of HuIFN-gamma also resulted in a substantial reduction in the ability of the molecule to be renatured in vitro from the treatment with chaotrophic agents, a method frequently used to extract and purify recombinant polypeptides from E. coli host. The latter result may account for some earlier reports which inferred the involvement of the COOH terminus in the functions of the HuIFN-gamma molecule due to their failure to detect activity upon deletions of only 11-18 residues.
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Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002872:Chromosome Deletion; D014644:Genetic Variation; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins; D015183:Restriction Mapping
D011994:Recombinant Proteins; D007371:Interferon-gamma