Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Chromatography

Background:

• Protein purification is crucial for high-throughput biological studies like proteomics and drug discovery.
• Current methods often struggle to meet the high yield, purity, and activity (HHH) requirements for structural and industrial applications.
• A single-step purification system achieving the HHH benchmark is highly desirable.

Purpose of the Study:

• To develop an efficient, single-step chromatographic technology for protein purification meeting the HHH benchmark.
• To leverage the ultra-high-affinity interaction between Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7).

Main Methods:

• Engineered a CL7 tag by mutating CE7, retaining binding affinity to Im7 while inactivating DNase activity.
• Developed a large-scale production and immobilization protocol for Im7 onto a solid support.
• Applied the CL7-Im7 system for one-step purification of diverse and challenging proteins.

Main Results:

• Demonstrated successful one-step HHH purification of various difficult-to-purify proteins, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins.
• The CL7-Im7 system exhibited ultra-high affinity (Kd ~ 10^-14-10^-17 M).
• The purification system is simple, reusable, and versatile.

Conclusions:

• The developed chromatographic technology offers an efficient solution for single-step protein purification meeting HHH standards.
• This system is applicable to a broad range of challenging biological molecules and can be used for pulldown and kinetic assays.
• The ultra-high affinity interaction provides a robust foundation for advanced biomolecular purification and analysis.