scholarly journals Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Fei Jin ◽  
Cheng Shen ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
False Negative ◽  
Membrane Topology ◽  
Size Exclusion ◽  
Expression Screening ◽  
Cell Extracts ◽  
Exclusion Chromatography

AbstractGFP 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.

scholarly journals Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

2020 ◽  
Author(s):  
Fei Jin ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
Motoyuki Hattori
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
Size Exclusion ◽  
Expression Screening ◽  
Functional Studies ◽  
Membrane Protein Expression ◽  
Exclusion Chromatography

AbstractMembrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. 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 and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

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