Protein stabilityIt is a key factor affecting its function, folding, and degradation, and directly measuring protein stability in living cells has always been a challenge in experimental research. Traditional methods are often complex to operate, time-consuming, and difficult to obtain quantitative results in live cells. The use of Intein based biosensors provides a new approach to address this issue.

The principle of peptide containing biosensors

Endopeptides are a type of self splicing peptide sequence that exists in host proteins. They can cleave themselves through self catalytic protein splicing reactions and reconnect the two end exons to form mature proteins. Due to its precise rearrangement ability of peptide bonds, intronic peptides have been widely used in protein engineering. Peptides contained in some microorganisms can also sense environmental stimuli such as metal, heat, and oxidative stress, serving as sensing elements for regulating protein function. Based on this, researchers have developed various splicing dependent reporting systems for screening splicing inhibitors or constructing biosensors.

Application of Peptide Containing Biosensors in Protein Stability Monitoring

In 2025, Christopher W. Lennon's team at Murray State University in the United States developed a peptide based biosensor for real-time monitoring of protein stability in living cells. The core of this method is to insert the target protein into the interior of the peptide, rather than directly fusing it to the reporter protein, effectively avoiding interference of the target protein on the structure and function of the reporter protein. Restoring the function of aminoglycoside resistance genes (KanR) through splicing directly correlates splicing activity with cell survival, facilitating the evaluation of protein stability through growth curves.

Specifically, this method utilizes the spontaneous splicing characteristics of endogenous peptides under specific conditions to directly link the stability of the target protein with splicing efficiency, achieving quantitative evaluation of protein stability in vivo. When the target protein is stable, the efficiency of peptide splicing is high, KanR function is restored, and cells survive in antibiotic containing culture medium; On the contrary, when the target protein is unstable, the efficiency of peptide splicing is low, KanR function cannot be restored, and cells die. By monitoring cell survival rate, the stability of the target protein can be indirectly evaluated.

Implementation of high-throughput analysis

This method has the potential for high-throughput analysis. By culturing cells with different mutants or under different treatment conditions separately in microplates, the protein stability of multiple samples can be monitored simultaneously. In addition, combining automated liquid processing systems and high-throughput sequencing technology can further improve analysis throughput.

Application prospects

Peptide based biosensors have broad application prospects in protein stability monitoring:

Rapid assessment of the impact of mutations on protein stability: By inserting the mutant into the interior of the peptide, key mutations that affect protein stability can be quickly screened.

Drug screening: By monitoring the changes in protein stability after drug treatment, the impact of drugs on protein function can be evaluated, providing important basis for drug development.

Optimization of chaperone protein expression: By monitoring the effect of chaperone protein expression on the stability of the target protein, the expression conditions of chaperone proteins can be optimized to improve the production efficiency of recombinant proteins.

Screening of small molecule compounds: By identifying small molecule compounds that affect protein stability or splicing reactions, their mechanisms of action can be explored, providing candidate compounds for new drug development.

Conclusion

Biosensors based on intrinsic peptides for living cellsprotein stabilityReal time monitoring provides a new paradigm. This method has the advantages of easy operation, high throughput, and accurate quantification, and has broad application prospects in protein stability research, drug screening, and recombinant protein production. With the continuous development of technology, peptide based biosensors are expected to play a greater role in life science research and the biopharmaceutical industry.