scholarly journals Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies

2014 ◽  
Vol 23 (9) ◽  
pp. 1309-1314 ◽  
Author(s):  
Joanne L. Parker ◽  
Simon Newstead
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Protein Expression ◽  
Functional Studies ◽  
Membrane Protein Expression ◽  
Eukaryotic Membrane Protein

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.

Membrane Protein Expression in Saccharomyces cerevisiae

2011 ◽  
pp. 37-73
Author(s):  
Zachary Britton ◽  
Carissa Young ◽  
Özge Can ◽  
Patrick McNeely ◽  
Andrea Naranjo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Protein Expression ◽  
Membrane Protein Expression

High‐throughput Cell‐free Screening of Eukaryotic Membrane Protein Expression in Lipidic Mimetics

2021 ◽  
Author(s):  
Renato Bruni ◽  
Aisha Laguerre ◽  
Anna‐Maria Kaminska ◽  
Sean McSweeney ◽  
Wayne A. Hendrickson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Expression ◽  
High Throughput ◽  
Membrane Protein Expression ◽  
Eukaryotic Membrane Protein

Quantitative Membrane Protein Expression at the Blood–Brain Barrier of Adult and Younger Cynomolgus Monkeys

2011 ◽  
Vol 100 (9) ◽  
pp. 3939-3950 ◽  
Author(s):  
Katsuaki Ito ◽  
Yasuo Uchida ◽  
Sumio Ohtsuki ◽  
Sanshiro Aizawa ◽  
Hirotaka Kawakami ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Expression ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Cynomolgus Monkeys ◽  
Membrane Protein Expression ◽  
Blood Brain
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