For immobilization of recombinant proteins on DNA nanostructures typically a high excess of proteins is used to drive the binding equilibrium in the favorable direction. For further experiments the unbound proteins have to be removed to prevent artefacts in bioassays.
The fractionation of DNA origami nanostructures with immobilized proteins and unbound proteins by FFE offers many advantages over conventional methods:
- Fast separation in ca. 15 minutes
- Biocompatible separation, preservation of protein activity
- No shear forces that can damage teritiary structures of proteins
- Prevention of protein aggregation
- Cooling of samples and buffers in separation chamber
- High recovery/reproducibility
- No limitation for size of DNA nanostructures and proteins
- Purification of analytical or quantitative amounts
For further information would like to direct attention to a first proof of concept with biotechnologically relevant enzymes P450 BM3 and GRE2, published in Angewandte Chemie Int. Ed.:
Assembly and Purification of Enzyme-Functionalized DNA Origami Structures. (DOI: 10.1002/anie.201500175)
In the future many other possible applications for separation of DNA nanostructures and heteroelements arise from these findings.
This work was done in cooperation with Prof. Dr. Christof M. Niemeyer, Karsruhe Institute of Technology.