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Updated: Nov 12, 2025
Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography
Published on: January 6, 2023
Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of
Fei Jin1, Cheng Shen1, Yao Wang1
1State Key Laboratory of Genetic Engineering, Shanghai Key Laboratory of Bioactive Small Molecules, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China.
Abstract:
GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.
