Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein...

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• PhaR protein, a repressor of polyhydroxyalkanoates (PHA) biosynthesis, binds to bacterial PHA granules.
• PhaR was engineered as an affinity tag for in vitro protein purification.
• Fusion of PhaR with a self-cleavable intein enables targeted protein release.

Purpose of the Study:

• To develop and validate a novel in vitro protein purification system utilizing PhaR affinity tags.
• To demonstrate the efficiency of the PhaR-based system for purifying various target proteins.

Main Methods:

• Constructing a fusion protein: PhaR-intein-target protein expressed in E. coli.
• Immobilizing the fusion protein onto PHA nanoparticles via PhaR binding.
• Inducing intein self-cleavage using a pH and temperature shift for protein release.
• Separating the purified target protein from PHA nanoparticles by centrifugation.

Main Results:

• Successfully expressed and purified fusion proteins in recombinant E. coli.
• Demonstrated efficient binding of PhaR-tagged proteins to PHA nanoparticles.
• Achieved successful purification of enhanced green fluorescent protein (EGFP), maltose binding protein (MBP), and beta-galactosidase (LacZ).

Conclusions:

• The PhaR-based system is a feasible and effective method for in vitro protein purification.
• The system offers a cost-effective alternative to commercial protein purification kits.
• Components are easily accessible, allowing researchers to establish their own purification strategies.