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.
Highlights
GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening
To overcome the disadvantages of the conventional FSEC method, we designed FSECNb, which utilizes short peptides as fusion tags and Nbs specific to these peptides fused to monomerized EGFP as a probe (Fig. 1)
Among the Zrt/Irt-like protein (ZIP) family, the structure of the bacterial ZIP protein from Bordetella bronchiseptica (BbZIP) was determined by crystallography[39]; we chose to utilize BbZIP to establish our FSEC-Nb system because both its N- and C-terminal ends are located at the periplasm[39], which is not well suited for application of the GFP fusion-based FSEC method in bacterial expression systems
Summary
GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. Following pioneering work on the application of GFP fusion techniques for membrane protein expression screening[12,13,14], GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely utilized for rapid evaluation of the expression status and thermostability of membrane proteins from both eukaryotes and prokaryotes[15,16]. This finding indicates that the simple strategy of N- or C-terminal GFP fusion is not applicable to some eukaryotic membrane proteins; the application of GFP fusion-based FSEC may require optimization of the position at which GFP is inserted To overcome such disadvantages, a GFP fusion-free FSEC method would be ideal, and a multivalent nitrilotriacetic acid (NTA) fluorescent probe called P3NTA was developed as a pioneering work of the GFP fusion-free FSEC method[9]. Such expression constructs would still be promising since structure determination by cryo-EM requires much less purified protein than structure determination by X-ray crystallography[34]
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