High expression in bacteria and purification of polymorphic mouse interleukin 2 molecules

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Six novel mouse interleukin 2 (IL-2) alleles, in addition to the known form, have been identified in different mouse strains.
• Studying the in vivo and in vitro biological activities of these diverse IL-2 alleles necessitates their production in large quantities.

Purpose of the Study:

• To develop a method for the large-scale production and purification of various mouse IL-2 alleles.
• To assess the biological activity of purified mouse IL-2 alleles using a cell-based assay.

Main Methods:

• Cloning of IL-2 cDNAs into a pET7-7/BL21(DE3) bacterial expression system for protein production.
• Purification of IL-2 using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and recovery of activity after SDS denaturation.
• Assaying the biological activity of purified IL-2 variants by supporting the growth of the IL-2-dependent cell line CTL-L2.

Main Results:

• Bacterial expression yielded 30-100 mg of IL-2 per litre of culture.
• Purified IL-2 molecules regained biological activity after SDS denaturation and removal of extraction reagents.
• All purified IL-2 alleles supported CTL-L2 cell growth, with the RF IL-2 allele (from NOD mice) exhibiting relatively lower activity.

Conclusions:

• A robust method was established for producing and purifying functional mouse IL-2 alleles from bacterial cultures.
• The purification strategy effectively recovers IL-2 activity, enabling further biological characterization of different alleles.
• Variations in biological activity among IL-2 alleles were observed, highlighting the importance of studying specific allelic forms.