Refolding proteins by gel filtration chromatography

Area of Science:

• Biochemistry
• Structural Biology
• Protein Chemistry

Background:

• Recombinant proteins are often produced as insoluble inclusion bodies, hindering their purification and structural analysis.
• Efficient refolding techniques are crucial for obtaining functional proteins for research and therapeutic applications.

Purpose of the Study:

• To develop a facile and general method for refolding milligram quantities of purified proteins.
• To demonstrate the applicability of the refolding technique to diverse proteins and macromolecular complexes.
• To enable structural analysis of refolded proteins using NMR spectroscopy and X-ray crystallography.

Main Methods:

• Gel filtration chromatography was employed as the primary technique for protein refolding.
• Electrophoretic mobility shift assays and NMR spectroscopy were used to confirm the native conformation of refolded proteins.

Main Results:

• Milligram quantities of human ETS-1 protein, bovine ribonuclease A, and E. coli integration host factor were successfully refolded into their native conformations.
• The technique was extended to prepare milligram quantities of macromolecular complexes.
• The method proved effective for proteins with diverse refolding challenges.

Conclusions:

• Gel filtration chromatography offers a general and facile approach for refolding purified proteins from insoluble inclusion bodies.
• This technique facilitates the preparation of sufficient quantities of native proteins and complexes for structural determination.
• The method has broad potential for recovering recombinant proteins for various applications.