An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Interleukin-7 (IL7) is crucial for T cell development but has a short half-life, limiting its therapeutic use.
• Chemical biotinylation often damages proteins, reducing their bioactivity.

Purpose of the Study:

• To develop a prokaryotic system for site-specific biotinylation of IL7 using the AviTag/BirA system.
• To compare the functional advantages of site-specific versus random biotinylation of IL7.

Main Methods:

• Constructed a recombinant plasmid for coexpressing AviTag-IL7 and BirA ligase in E. coli.
• Optimized protein expression and purification using Ni-NTA chromatography.
• Assessed biotinylation efficiency via Western blot and PAGE; evaluated IL7 function through T cell proliferation and apoptosis assays.

Main Results:

• Successfully produced site-specifically biotinylated IL7 (biotin-IL7) with retained T cell proliferative activity.
• Site-specific biotinylation demonstrated superior T cell proliferation induction compared to random biotinylation.
• Site-specific biotin-IL7 showed reduced interference with antibody binding and enhanced apoptosis downregulation.

Conclusions:

• The AviTag/BirA system provides an efficient, scalable method for producing functional biotin-IL7.
• Site-specific biotinylation preserves IL7 bioactivity and enhances therapeutic potential, particularly for cancer immunotherapy.
• Further optimization of soluble expression and biotinylation efficiency is warranted.