High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices.

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Biochemistry

Background:

• Mammalian cell lines are crucial for expressing complex proteins with native post-translational modifications.
• Low transfection efficiency is a significant hurdle in utilizing mammalian expression systems.
• Fluorescent protein fusions aid in monitoring expression and facilitate protein purification.

Purpose of the Study:

• To develop and validate nanobody affinity matrices for purifying GFP/YFP and mCherry fusion proteins.
• To elucidate the binding mechanisms of nanobodies to target proteins via X-ray crystallography.
• To establish an efficient and robust system for recombinant protein expression and purification in mammalian cells.

Main Methods:

• Development of anti-GFP and anti-mCherry nanobody affinity matrices.
• Purification of GFP/YFP and mCherry tagged recombinant proteins.
• X-ray crystallography to determine protein-nanobody binding interactions.
• Stability testing of nanobody-sepharose supports under various conditions.
• Application of the mCherry-tag system for purifying human topoisomerase 2α in HEK293F cells.

Main Results:

• A panel of anti-GFP and anti-mCherry nanobody affinity matrices were evaluated for purification efficacy.
• X-ray crystallography provided molecular insights into nanobody-protein binding.
• An optimal nanobody pair was identified for purifying GFP/YFP or mCherry tagged proteins.
• Nanobody-sepharose supports demonstrated stability through multiple cleaning cycles and denaturing conditions.
• Successful purification of recombinant human topoisomerase 2α using the mCherry-tag system was achieved.

Conclusions:

• Developed nanobody affinity matrices offer a high-efficiency method for purifying fluorescently tagged proteins in mammalian systems.
• The established systems allow for real-time monitoring of protein expression and simplified purification.
• These tools are valuable for both individual and tandem applications in mammalian protein expression.