A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC)

Area of Science:

• Molecular Biology
• Biotechnology

Background:

• Current protein-protein interaction techniques often suffer from variable fusion protein expression levels due to the use of multiple plasmids.
• This variability can compromise the accuracy and reproducibility of experimental results.

Purpose of the Study:

• To introduce a novel recombination-based cloning strategy, named 2in1, for the co-expression of fusion proteins from a single plasmid.
• To develop an improved ratiometric bimolecular fluorescence complementation assay (rBiFC) utilizing the 2in1 strategy for more reliable protein interaction analysis.

Main Methods:

• Developed the 2in1 strategy for single-plasmid co-expression of fusion proteins using recombination-based cloning.
• Engineered a ratiometric bimolecular fluorescence complementation assay (rBiFC) by integrating the 2in1 strategy into a single vector with an internal fluorescent marker.

Main Results:

• The 2in1 strategy enables consistent cell-by-cell co-expression of fusion proteins, overcoming expression variability issues.
• The developed rBiFC assay demonstrated increased credibility of protein-protein interaction results through ratiometric comparisons.
• The 2in1 strategy proved feasible for rBiFC and adaptable to other multi-gene expression vector systems.

Conclusions:

• The 2in1 cloning strategy offers a robust solution for co-expressing multiple genes, particularly fusion proteins, from a single plasmid.
• This approach significantly enhances the reliability and interpretability of protein-protein interaction studies, such as rBiFC.
• The 2in1 strategy holds potential for future applications in synthetic biology and other multi-gene expression systems.