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Updated: Jun 16, 2026
Site-Specific Lysine Lactylation via Genetic Code Expansion in E. coli and Mammalian Cells
Published on: February 24, 2026
Creation of a lysine-deficient LIGHT mutant with the capacity for site-specific PEGylation and low affinity for a
Tomohiro Morishige1, Yasuo Yoshioka, Hiroshi Inakura
1Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Abstract:
The cytokine LIGHT is a promising candidate for cancer therapy. However, the therapeutic effect of LIGHT as a systemic anticancer agent is currently insufficient because of its instability and its binding to nonfunctional soluble decoy receptor 3 (DcR3), which is overexpressed in various tumors. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation may occur randomly at all lysine residues and the NH(2)-terminus; therefore, PEGylated proteins are generally heterogeneous and have decreased bioactivity. In this study, we attempted to create a lysine-deficient LIGHT mutant that could be PEGylated site-specifically and would have lower affinity for DcR3. We prepared phage libraries expressing LIGHT mutants in which all the lysine residues were replaced with other amino acids. A lysine-deficient LIGHT mutant [mLIGHT-Lys(-)] was isolated by panning against lymphotoxin beta receptor (LTbetaR). mLIGHT-Lys(-) could be site-specifically PEGylated at its NH(2)-terminus, yielding molecular uniformity and in vitro bioactivity equal to that of non-PEGylated, wild-type LIGHT. Furthermore, mLIGHT-Lys(-) was not trapped by the nonfunctional DcR3, despite binding to its functional receptors. These results suggest that mLIGHT-Lys(-) might be a useful candidate for cancer therapy.
Insights
Engineered cytokine LIGHT, a potential cancer therapy, shows improved stability and reduced binding to decoy receptor 3 through site-specific PEGylation of a lysine-deficient mutant, enhancing its therapeutic potential.
Area of Science:
- Biotechnology
- Immunology
- Cancer Research
Background:
- Cytokine LIGHT shows promise for cancer therapy but suffers from instability and binding to decoy receptor 3 (DcR3).
- Standard PEGylation improves protein stability but often results in heterogeneous products with reduced bioactivity.
Purpose of the Study:
- To develop a modified LIGHT cytokine with enhanced stability and reduced affinity for DcR3 for improved cancer therapy.
- To create a site-specifically PEGylatable LIGHT mutant with preserved bioactivity.
Main Methods:
- Generated phage libraries of LIGHT mutants with all lysine residues replaced.
- Isolated a lysine-deficient LIGHT mutant (mLIGHT-Lys(-)) via panning against lymphotoxin beta receptor (LTbetaR).
- Performed site-specific N-terminal PEGylation on the mLIGHT-Lys(-) mutant.
Main Results:
- The lysine-deficient LIGHT mutant (mLIGHT-Lys(-)) was successfully created and could be site-specifically PEGylated at the N-terminus.
- PEGylated mLIGHT-Lys(-) exhibited molecular uniformity and in vitro bioactivity comparable to wild-type LIGHT.
- mLIGHT-Lys(-) demonstrated reduced trapping by soluble DcR3 while retaining binding to functional receptors.
Conclusions:
- Site-specific PEGylation of a lysine-deficient LIGHT mutant yields a stable, homogeneous therapeutic agent.
- The modified LIGHT mutant (mLIGHT-Lys(-)) overcomes limitations of instability and decoy receptor binding, suggesting its potential as a cancer therapeutic.
