Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• High-yield recombinant protein expression is crucial for structural biology techniques like X-ray crystallography and cryo-electron microscopy.
• Cultured human cells offer ideal machinery for expressing complex human proteins but present technical challenges compared to bacterial systems.
• Efficient production of pure proteins and multi-protein complexes requires optimized expression systems.

Purpose of the Study:

• To develop an efficient recombinant protein expression system using cultured human cells.
• To overcome technical challenges associated with human cell culture for protein expression.
• To enable high-yield production of pure recombinant human proteins for structural biology.

Main Methods:

• Utilized a Yellow Fluorescent Protein (YFP) fusion-tag for recombinant protein generation in suspension-cultured HEK293F cells.
• Employed YFP for direct visualization and fluorescence-based selection of high-expressing clones.
• Developed a rapid purification protocol using anti-GFP/YFP nanobody support.

Main Results:

• Demonstrated successful expression of large human proteins, TOP2α (340 KDa dimer) and TOP2β catalytic core (260 KDa dimer).
• Achieved robust and reproducible yields of >10 mg/L using transient expression.
• Obtained yields of 2.5 mg/L using stable expression.

Conclusions:

• The YFP fusion-tag system provides an efficient method for producing high yields of pure recombinant human proteins in HEK293F cells.
• This system facilitates the selection and purification of proteins for structural biology.
• The developed method addresses challenges in human cell-based protein expression, offering a valuable tool for researchers.