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Structure-function analysis of the human interferon gamma. The COOH terminus is not essential for functional activity
1Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.
The Journal of Biological Chemistry
|August 5, 1990
Summary
The carboxyl-terminal 21 residues of human interferon gamma (HuIFN-gamma) are not essential for its antiviral or antiproliferative functions. However, these residues are important for protein stability and in vitro renaturation, explaining previous findings.
Area of Science:
- Molecular Biology
- Immunology
- Protein Engineering
Background:
- Human interferon gamma (HuIFN-gamma) is a crucial cytokine with antiviral and antiproliferative properties.
- Understanding the structure-function relationship of HuIFN-gamma is vital for its therapeutic applications.
- Previous studies suggested the involvement of the carboxyl terminus in HuIFN-gamma function.
Purpose of the Study:
- To investigate the role of the carboxyl-terminal residues in the structure and function of HuIFN-gamma.
- To elucidate the impact of COOH-terminal deletions on HuIFN-gamma's biological activities and stability.
Main Methods:
- Construction and expression of four recombinant HuIFN-gamma variants with varying COOH-terminal deletions (122, 117, 111, 106 amino acids) in Escherichia coli.
- Assessment of antiviral and antiproliferative activities of the variants.
- Evaluation of intracellular accumulation levels and in vitro transcription-translation efficiency.
- Analysis of in vitro renaturation capabilities using chaotrophic agents.
Main Results:
- Deletion of the terminal 21 amino acid residues caused only minor reductions (2-3 fold) in antiviral and antiproliferative activities.
- Further deletions (26, 32, 37 residues) resulted in undetectable activity and significantly reduced intracellular accumulation (50-100 fold).
- In vitro transcription-translation assays showed comparable production efficiency for all variants, suggesting instability of truncated forms in E. coli.
- Loss of the terminal 21 residues substantially impaired in vitro renaturation of HuIFN-gamma.
Conclusions:
- The terminal 21 amino acid residues of HuIFN-gamma are not directly involved in its core biological functions.
- These residues play a critical role in protein stability and facilitate proper refolding, which may explain prior conflicting results.
- The findings provide insights into the structural requirements for functional and stable HuIFN-gamma.