[Human tumor necrosis factor mutants: preparation and some properties]

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Human tumor necrosis factor-alpha (TNF-alpha) is a key cytokine in inflammation and immunity.
• Understanding TNF-alpha's structure-function relationship is crucial for developing targeted therapies.

Purpose of the Study:

• To generate and characterize novel TNF-alpha muteins with specific amino acid substitutions and deletions.
• To investigate the impact of these mutations on TNF-alpha's structure, stability, and biological activity.

Main Methods:

• Polymerase chain reaction (PCR) was used to create mutant genes encoding TNF-alpha.
• Mutant proteins (muteins) were expressed in E. coli and purified using a novel method.
• Circular dichroism (CD) and cross-linking assays assessed protein structure.
• Cytotoxicity assays evaluated the biological activity of the muteins.

Main Results:

• Several TNF-alpha muteins with point mutations (R32H, A33S, F144L, I118M, I118A) and a deletion (residues 67-70) were successfully produced.
• Most muteins exhibited spatial structures similar to native TNF-alpha, as confirmed by CD and cross-linking.
• Mutants R32H and A33S showed decreased cytotoxicity against L929 cells.
• Mutants F144L and R32H-F144L were found to be essentially inactive.

Conclusions:

• Specific mutations in TNF-alpha can significantly alter its biological activity while maintaining structural integrity.
• The F144L mutation and the R32H-F144L double mutation abolish TNF-alpha cytotoxicity.
• These findings provide valuable structure-activity relationship data for TNF-alpha, relevant for therapeutic development.