Use Intein Cleavable Polyhydroxyalkanoate Synthase Fusions to Improve Protein Solubility

Area of Science:

• Biotechnology
• Protein Purification
• Metabolic Engineering

Background:

• Polyhydroxyalkanoates (PHAs) are biopolyesters with diverse applications.
• Efficient purification of recombinant proteins is crucial for biotechnology and pharmaceuticals.
• Intein self-cleaving technology offers a method for protein purification.

Purpose of the Study:

• To develop a novel bioseparation resin using PHA particles for efficient protein purification.
• To demonstrate the release of tagless, soluble target proteins from PHA particles.
• To evaluate the cost-effectiveness and versatility of this purification method.

Main Methods:

• Recombinant E. coli were engineered to produce intein-cleavable PHA synthase fusions.
• Intracellular PHA particles formed were coated with target protein fusions.
• PHA particles were purified from lysed cells.
• Target proteins were released by pH reduction.

Main Results:

• PHA particles efficiently purified intracellularly formed protein fusions.
• Tagless, soluble target proteins were released with pharmaceutical-grade purity.
• The method was successfully applied to purify six diverse target proteins, including GFP, Rv1626, ZZ domain, TNFα, G-CSF, and IFNα2b.
• This method provides a cost-effective alternative to commercial affinity separation technologies.

Conclusions:

• PHA particles can serve as an effective bioseparation resin for purifying soluble target proteins.
• This intein-mediated approach offers a simplified, cost-effective purification strategy.
• The technique demonstrates broad applicability for various recombinant proteins.