Effective expression and purification of bioactive recombinant soluble LIGHT

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• LIGHT (TNFSF14, CD258) is a TNF superfamily member crucial for immune responses and cell regulation.
• Soluble forms of LIGHT are important for its biological functions, but production challenges exist.
• Previous studies faced difficulties in producing bioactive soluble mouse LIGHT (s-mLIGHT).

Purpose of the Study:

• To establish effective strategies for producing bioactive soluble human LIGHT (s-hLIGHT) and mouse LIGHT (s-mLIGHT).
• To investigate the role of trimerization in the biological activity of s-mLIGHT.

Main Methods:

• Optimized refolding of s-hLIGHT from Escherichia coli inclusion bodies using L-cysteine.
• Engineered trimeric s-mLIGHT (Foldon-mLIGHT) by fusing it with the T4 fibritin 'foldon' domain.
• Assessed the biological activity of Foldon-mLIGHT on mouse carcinoma cells.

Main Results:

• L-cysteine addition significantly enhanced s-hLIGHT refolding efficiency.
• Trimeric Foldon-mLIGHT demonstrated potent inhibition of mouse carcinoma cell growth in the picomolar range.
• Trimerization was confirmed as essential for s-mLIGHT's biological activity.

Conclusions:

• Effective methods for producing bioactive s-hLIGHT and trimeric s-mLIGHT were established.
• Trimerization is critical for the potent anti-cancer activity of soluble mouse LIGHT.
• This work facilitates further research into LIGHT's biological roles and therapeutic potential.