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

Publication Type:

Journal Article


Protein Sci, Volume 31, Issue 9, p.e4383 (2022)


Animals, Chromatography, Affinity, Crystallography, X-Ray, Humans, Mammals, Recombinant Fusion Proteins, Recombinant Proteins, Single-Domain Antibodies


<p>Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein&#39;s native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.</p>

7SAH (LaG16-eGFP complex), 7SAI (Lag30-eGFP complex), 7SAJ (LaM2-mCherry complex), 7SAK (LaM4-mCherry complex), and 7SAL (LaM6 complex)