Evolution of interleukin-15 for higher E. coli expression and solubility.

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Interleukin-15 (IL-15) is crucial for immune cell proliferation.
• Improving IL-15 solubility and expression in Escherichia coli presents a challenge for recombinant protein production.

Purpose of the Study:

• To engineer IL-15 variants with enhanced solubility and cytoplasmic over-expression in E. coli.
• To characterize the binding affinity and biological activity of the improved IL-15 mutants.

Main Methods:

• Directed evolution coupled with a protein solubility selection system using chloramphenicol acetyltransferase (CAT) reporter.
• Expression of IL-15 as an N-terminal fusion to CAT in E. coli.
• ELISA screening to assess IL-15-CAT fusion binding to IL-15 receptor alpha (IL-15Rα) Sushi domain.
• Characterization of IL-15 mutant binding and T-cell growth stimulation assays.

Main Results:

• Two IL-15 variants, M38 and M253, were identified with significantly improved solubility (12- to 18-fold increase).
• M38 demonstrated binding affinity to IL-15Rα comparable to wild-type (WT) IL-15.
• Both M38 and M253 effectively stimulated T-cell growth, acting as agonists of IL-15.

Conclusions:

• Directed evolution successfully generated highly soluble and over-expressed IL-15 mutants in E. coli.
• Engineered IL-15 variants M38 and M253 retain potent IL-15Rα binding and biological activity.
• These improved IL-15 variants hold promise for enhanced therapeutic applications.