scholarly journals Membrane protein extraction and purification using styrene–maleic acid (SMA) copolymer: effect of variations in polymer structure

2016 ◽  
Vol 473 (23) ◽  
pp. 4349-4360 ◽  
Author(s):  
Kerrie A. Morrison ◽  
Aneel Akram ◽  
Ashlyn Mathews ◽  
Zoeya A. Khan ◽  
Jaimin H. Patel ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Maleic Acid ◽  
Protein Extraction ◽  
Affinity Purification ◽  
Expression System ◽  
Transmembrane Proteins ◽  
Membrane Extraction ◽  
Total Size ◽  
Average Molecular Mass

The use of styrene–maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5–10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

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 Selective Enrichment of Membrane Proteins by Partition Phase Separation for Proteomic Studies

2003 ◽  
Vol 2003 (4) ◽  
pp. 249-255 ◽  
Author(s):  
M. Walid Qoronfleh ◽  
Betsy Benton ◽  
Ray Ignacio ◽  
Barbara Kaboord
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Extraction ◽  
Yeast Cells ◽  
Protein Markers ◽  
Two Phase ◽  
Western Blots ◽  
Selective Enrichment ◽  
Phase Partitioning ◽  
Hydrophobic Fraction

The human proteome project will demand faster, easier, and more reliable methods to isolate and purify protein targets. Membrane proteins are the most valuable group of proteins since they are the target for 70–80% of all drugs. Perbio Science has developed a protocol for the quick, easy, and reproducible isolation of integral membrane proteins from eukaryotic cells. This procedure utilizes a proprietary formulation to facilitate cell membrane disruption in a mild, nondenaturing environment and efficiently solubilizes membrane proteins. The technique utilizes a two-phase partitioning system that enables the class separation of hydrophobic and hydrophilic proteins. A variety of protein markers were used to investigate the partitioning efficiency of the membrane protein extraction reagents (Mem-PER) (Mem-PER is a registered trademark of Pierce Biotechnology, Inc) system. These included membrane proteins with one or more transmembrane spanning domains as well as peripheral and cytosolic proteins. Based on densitometry analyses of our Western blots, we obtained excellent solubilization of membrane proteins with less than 10% contamination of the hydrophobic fraction with hydrophilic proteins. Compared to other methodologies for membrane protein solubilization that use time-consuming protocols or expensive and cumbersome instrumentation, the Mem-PER reagents system for eukaryotic membrane protein extraction offers an easy, efficient, and reproducible method to isolate membrane proteins from mammalian and yeast cells.

scholarly journals Membrane-active Polymers: NCMNP13-x, NCMNP21-x and NCMNP21b-x for Membrane Protein Structural Biology

2022 ◽  
Author(s):  
Thi Kim Hoang Trinh ◽  
Claudio Catalano ◽  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Maleic Acid ◽  
Drug Targets ◽  
Design Synthesis ◽  
Native Cell ◽  
Wide Range ◽  
Lipid Particles ◽  
High Resolution Structure

Membrane proteins are a ubiquitous group of bio-macromolecules responsible for many crucial biological processes and serve as drug targets for a wide range of modern drugs. Detergent-free technologies such as styrene-maleic acid lipid particles (SMALP), diisobutylene-maleic acid lipid particles (DIBMALP), and native cell membrane nanoparticles (NCMN) systems have recently emerged as revolutionary alternatives to the traditional detergent-based approaches for membrane protein research. NCMN systems aim to create a membrane-active polymer library suitable for high-resolution structure determination. Herein, we report our design, synthesis, characterization and comparative application analyses of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x and NCMNP21b-x. Although each NCMN polymer can solubilize various model membrane proteins and conserve native lipids into NCMN particles, only the NCMNP21b-x series reveals lipid-protein particles with good buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. Consequently, the NCMNP21b-x polymers that bring out high-quality NCMN particles are particularly attractive for membrane protein structural biology.

Surfactant-free purification of membrane proteins with intact native membrane environment

2011 ◽  
Vol 39 (3) ◽  
pp. 813-818 ◽  
Author(s):  
Mohammed Jamshad ◽  
Yu-Pin Lin ◽  
Timothy J. Knowles ◽  
Rosemary A. Parslow ◽  
Craig Harris ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Maleic Acid ◽  
Biochemical Study ◽  
Soluble Proteins ◽  
Structure And Function ◽  
Three Dimensions ◽  
Present Review ◽  
Protein Particles ◽  
And Function

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

scholarly journals Lipidomic and in-gel analysis of maleic acid co-polymer nanodiscs reveals differences in composition of solubilized membranes

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Marta Barniol-Xicota ◽  
Steven H. L. Verhelst
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Content ◽  
Lipid Bilayer ◽  
Maleic Acid ◽  
Chemical Properties ◽  
Aqueous Buffer ◽  
Detergent Solubilization

AbstractMembrane proteins are key in a large number of physiological and pathological processes. Their study often involves a prior detergent solubilization step, which strips away the membrane and can jeopardize membrane protein integrity. A recent alternative to detergents encompasses maleic acid based copolymers (xMAs), which disrupt the lipid bilayer and form lipid protein nanodiscs (xMALPs) soluble in aqueous buffer. Although xMALPs are often referred to as native nanodiscs, little is known about the resemblance of their lipid and protein content to the native bilayer. Here we have analyzed prokaryotic and eukaryotic xMALPs using lipidomics and in-gel analysis. Our results show that the xMALPs content varies with the chemical properties of the used xMA.

Comparative Analysis of Maleic Acid Copolymer-Based Lipid Nanodiscs Reveals Preferential Lipid and Protein Solubilization

2020 ◽  
Author(s):  
Marta Barniol-Xicota ◽  
Steven Verhelst
Keyword(s):  
Comparative Analysis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Content ◽  
Lipid Bilayer ◽  
Maleic Acid ◽  
Chemical Properties ◽  
Acid Copolymer ◽  
Lipid Nanodiscs ◽  
Maleic Acid Copolymer

Membrane proteins are key in a large number of physiological and pathological processes. Their study often involves a prior detergent solubilization step, which strips away the membrane and can jeopardize membrane protein integrity. A recent alternative to detergents encompasses maleic acid based copolymers (xMAs), which disrupt the lipid bilayer and form lipid protein nanodiscs (xMALPs) soluble in aqueous buffer. Although xMALPs are often referred to as native nanodiscs, little is known about the resemblance of their lipid and protein content to the native bilayer. Here we have analyzed prokaryotic and eukaryotic xMALPs using lipidomics and in-gel analysis. Our results show that the xMALPs content varies with the chemical properties of the used xMA and that some of these nanodiscs are less native than initially thought.<br>

scholarly journals Membrane Protein Extraction by Styrene-Maleic Acid Copolymers is Independent of Subcellular Localization but is Influenced by Membrane Organization

2017 ◽  
Vol 112 (3) ◽  
pp. 385a
Author(s):  
Jonas M. Doerr ◽  
Juan J. Dominguez Pardo ◽  
Marleen H. van Coevoorden-Hameete ◽  
Casper C. Hoogenraad ◽  
J. Antoinette Killian
Keyword(s):  
Membrane Protein ◽  
Subcellular Localization ◽  
Maleic Acid ◽  
Protein Extraction ◽  
Membrane Organization

scholarly journals Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform

2017 ◽  
Author(s):  
Shaobin Guo ◽  
Amit Vaish ◽  
Qing Chen ◽  
Richard M. Murray
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Specific Binding ◽  
Expression System ◽  
Point Of View ◽  
Biologically Active ◽  
Free Expression ◽  
Single Chain ◽  
Active Membrane ◽  
Free Environment

AbstractCell-free transcription-translation platforms have been widely utilized to express soluble proteins in basic synthetic biological circuit prototyping. From a synthetic biology point of view, it is critical to express membrane proteins in cell-free transcription-translation systems, and use them directly in biocircuits, considering the fact that histidine kinases, G-protein coupled receptors (GPCRs) and other important biosensors are all membrane proteins. Previous studies have expressed membrane proteins in cell-free systems with the help of detergents, liposomes or nanodiscs, but have not demonstrated the ability to prototype circuit behavior for the purpose of testing more complex circuit functions involving membrane-bound proteins. Built on previous efforts, in this work we demonstrated that we could co-translationally express solubilized and active membrane proteins in our cell-free TX-TL platform with membrane-like materials. We first tested the expression of several constructs with β1 and β2 adrenergic receptors in TX-TL and observed significant insoluble membrane protein production. The addition of nanodiscs to the cell free expression system enabled solubilization of membrane proteins. Nanodisc is lipoprotein-based membrane-like material. The activity of β2 adrenergic receptor was tested with both fluorescence and Surface Plasmon Resonance (SPR) binding assays by monitoring the specific binding response of small-molecule binders, carazolol and norepinephrine. Our results suggest that it is promising to use cell-free expression systems to prototype synthetic biocircuits involving single chain membrane proteins without extra procedures. This data made us one step closer to testing complex membrane protein circuits in cell-free environment.

scholarly journals Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs

2018 ◽  
Vol 54 (97) ◽  
pp. 13702-13705 ◽  
Author(s):  
Nils Hellwig ◽  
Oliver Peetz ◽  
Zainab Ahdash ◽  
Igor Tascón ◽  
Paula J. Booth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Maleic Acid ◽  
Protein Complexes ◽  
Native Mass Spectrometry ◽  
Direct Extraction ◽  
Membrane Protein Complexes

Other than more widely used methods, the use of styrene maleic acid copolymers allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding.

Artificial stabilization of the fusion pore by intra-organelle styrene-maleic acid copolymers

Soft Matter ◽  
2021 ◽  
Author(s):  
Marcelo Caparotta ◽  
Marcelo Puiatti ◽  
Diego Masone
Keyword(s):  
Membrane Protein ◽  
Maleic Acid ◽  
Protein Extraction ◽  
Fusion Pore ◽  
Protein Isolation ◽  
Free Alternative ◽  
Extraction And Purification ◽  
Lipid Environment

Styrene-maleic acid copolymers have become an advantageous detergent-free alternative for membrane protein isolation. Since their discovery, experimental membrane protein extraction and purification by keeping intact their lipid environment has become...

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