Fluorescence-detection Size-exclusion Chromatography Utilizing Nanobody Technology for Expression Screening of Membrane Proteins

Overview

Journal Commun Biol

Specialty Biology

Date 2021 Mar 20

PMID 33742097

Citations 9

Authors

Fei Jin

Cheng Shen

Yao Wang

Mengqi Wang

Minxuan Sun

Motoyuki Hattori

Affiliations

Soon will be listed here.

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.

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