Database Study on the Expression and Purification of Membrane Proteins

2021 ◽  
Vol 28 ◽  
Author(s):  
Chen-Yan china Zhang ◽  
Shi-Qi Zhao ◽  
Shi-Long Zhang ◽  
Li-Heng Luo ◽  
Ding-Chang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Drug Targets ◽  
Expression System ◽  
Data Bank ◽  
Hek293 Cells ◽  
Expression And Purification ◽  
Beta Barrel ◽  
Membrane Protein Expression

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

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.

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

scholarly journals Sex Hormones Regulate Rat Hepatic Monocarboxylate Transporter Expression and Membrane Trafficking

2017 ◽  
Vol 20 (1) ◽  
pp. 435 ◽  
Author(s):  
Jieyun Cao ◽  
Michael Ng ◽  
Melanie A Felmlee
Keyword(s):  
Sex Differences ◽  
Membrane Protein ◽  
Protein Expression ◽  
Estrous Cycle ◽  
Sex Hormones ◽  
Membrane Trafficking ◽  
Drug Disposition ◽  
Membrane Expression ◽  
Membrane Protein Expression ◽  
Estrous Cycle Stage

Purpose: Monocarboxylate transporters (MCTs) are involved in the transport of monocarboxylates such as ketone bodies, lactate, and pharmaceutical agents. CD147 functions as an ancillary protein for MCT1 and MCT4 for plasma membrane trafficking. Sex differences in MCT1 and MCT4 have been observed in muscle and reproductive tissues; however, there is a paucity of information on MCT sex differences in tissues involved in drug disposition. The objective of the present study was to quantify hepatic MCT1, MCT4 and CD147 mRNA, total cellular and membrane protein expression in males, over the estrous cycle in females and in ovariectomized (OVX) females. Method: Liver samples were collected from females at the four estrous cycle stages (proestrus, estrus, metestrus, diestrus), OVX females and male Sprague-Dawley rats (N = 3 – 5). Estrus cycle stage of females was determined by vaginal lavage. mRNA and protein (total and membrane) expression of MCT1, MCT4 and CD147 was evaluated by qPCR and western blot analysis. Results: MCT1 mRNA and membrane protein expression varied with estrous cycle stage, with OVX females having higher expression than males, indicating that female sex hormones may play a role in MCT1 regulation. MCT4 membrane expression varied with estrous cycle stage with expression significantly lower than males. MCT4 membrane expression in OVX females was also lower than males, suggesting that androgens play a role in membrane expression of MCT4. Males had higher membrane CD147 expression, whereas there was no difference in whole cell protein and mRNA levels suggesting that androgens are involved in regulating CD147 membrane localization. Conclusions: This study demonstrates hepatic expression and membrane localization of MCT1, MCT4 and CD147 are regulated by sex hormones. Sex differences in hepatic MCT expression may lead to altered drug disposition, so it is critical to elucidate the underlying mechanisms in the sex hormone-dependent regulation of MCT expression. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

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