Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Enhanced green fluorescent protein (EGFP) is a widely used reporter protein.
• Traditional EGFP expression can be limited by factors like protein folding and aggregation.
• Fragment-based approaches offer alternative strategies for protein detection and imaging.

Purpose of the Study:

• To establish a protocol for isolating profluorescent EGFP fragments.
• To enable rapid fluorescence reconstitution through fragment complementation.
• To provide a method for efficient purification and conjugation of EGFP fragments.

Main Methods:

• Cloning and overexpression of EGFP fragments fused to self-splitting intein in E. coli.
• Solubilization and refolding of fusion proteins from inclusion bodies.
• Purification of cleaved EGFP fragments using chitin affinity chromatography.
• Complementation of purified EGFP fragments with complementary oligonucleotides.

Main Results:

• Isolation of EGFP N-terminal fragment with a preformed chromophore.
• Demonstration of rapid and bright fluorescence upon complementation of EGFP fragments.
• Successful purification of EGFP fragments from recombinant E. coli.
• Protocol completion within approximately 3 days for fragment isolation and 1-4 hours for conjugation.

Conclusions:

• The developed protocol allows for the isolation of functional, profluorescent EGFP fragments.
• Fragment complementation provides a rapid method for generating fluorescence.
• This technique offers a versatile tool for applications requiring fast and efficient fluorescent protein generation.