Purification Of Membrane Proteins Research Articles

Conformational transitions of maleic acid segment drive pH induced changes in SMA polymer structure and solubility

The popularity of styrene-maleic acid (SMA) copolymers is based on their ability to solubilize biological membranes into nanoscale disks facilitating the extraction and purification of membrane proteins. Unfortunately, these disks are unstable in acidic environments which is due to the conformational transition of the SMA polymer. In this work, using a combination of NMR, IR and fluorescence spectroscopy together with DLS and quantum chemical calculations, we have investigated the pH-mediated conformational transition in the SMA polymer. With decreasing pH, polymer undergoes the following changes: 1) the dihedral angle in the maleic moieties of the polymer changes from 180° to 60° and then reaches 50°; 2) the carboxyl groups of the maleic fragment form hydrogen bonds with other maleic fragments, leading to aggregation of the SMA molecules; 3) the phenyl rings of the styrene fragments are brought closer together and the hydrophobicity of the environment inside the SMA polymer globule increases.

Unveiling the Relationship between Ceftobiprole and High-Molecular-Mass (HMM) Penicillin-Binding Proteins (PBPs) in Enterococcus faecalis.

Low-affinity PBP4, historically linked to penicillin resistance in Enterococcus faecalis, may still have affinity for novel cephalosporins. Ceftobiprole (BPR) is a common therapeutic choice, even with PBP4-related overexpression and amino acid substitution due to mutations. Our study aims to explore the interaction between BPR and High-Molecular-Mass (HMM) low-reactive PBPs in Penicillin-Resistant-Ampicillin-Susceptible/Ceftobiprole Non-Susceptible (PRAS/BPR-NS) E. faecalis clinical isolates. We conducted competition assays examining class A and B HMM PBPs from four PRAS/BPR-NS E. faecalis strains using purified membrane proteins and fluorescent penicillin (Bocillin FL), in treated and untreated conditions. Interaction strength was assessed calculating the 50% inhibitory concentration (IC50) values for ceftobiprole, by analyzing fluorescence intensity trends. Due to its low affinity, PBP4 did not display significant acylation among all strains. Moreover, both PBP1a and PBP1b showed a similar insensitivity trend. Conversely, other PBPs showed IC50 values ranging from 1/2-fold to 4-fold MICs. Upon higher BPR concentrations, increased percentages of PBP4 inhibition were observed in all strains. Our results support the hypothesis that PBP4 is necessary but not sufficient for BPR resistance, changing the paradigm for enterococcal cephalosporin resistance. We hypothesize that cooperation between class B PBP4 and at least one bifunctional class A PBP could be required to synthesize peptidoglycan and promote growth.

Lysosomal membrane transporter purification and reconstitution for functional studies.

Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

Selective extraction of recombinant membrane proteins from Hansenula polymorpha by pulsed electric field and lytic enzyme pretreatment

BackgroundIn yeast, recombinant membrane proteins including viral scaffold proteins used for the formation of enveloped Virus-like particles (eVLPs) typically accumulate intracellularly. Their recovery is carried out by mechanical disruption of the cells, often in combination with detergent treatment. Cell permeabilization is an attractive alternative to mechanical lysis because it allows for milder and more selective recovery of different intracellular products.ResultsHere, we present a novel approach for extraction of integral membrane proteins from yeast based on cell envelope permeabilization through a combination of pulsed electric field and lytic enzyme pretreatment of the cells. Our primary experiments focused on Hansenula polymorpha strain #25-5 co-expressing the integral membrane small surface protein (dS) of the duck hepatitis B virus and a fusion protein of dS with a trimer of a Human papillomavirus (HPV) L2-peptide (3xL2-dS). Irreversible plasma membrane permeabilization was induced by treating the cell suspension with monopolar rectangular pulses using a continuous flow system. The permeabilized cells were incubated with lyticase and dithiothreitol. This treatment increased the cell wall permeability, resulting in the release of over 50% of the soluble host proteins without causing significant cell lysis. The subsequent incubation with Triton X-100 resulted in the solubilization and release of a significant portion of 3xL2-dS and dS from the cells. By applying two steps: (i) brief heating of the cells before detergent treatment, and (ii) incubation of the extracts with KSCN, an 80% purity on the protein level has been achieved. Experiments performed with H. polymorpha strain T#3-3, co-expressing dS and the fusion protein EDIIIWNV-dS consisting of dS and the antigen from the West Nile virus (WSV), confirmed the applicability of this approach for recovering dS. The treatment, optimal for solubilization of 3xL2-dS and a significant part of dS, was not effective in isolating the fused protein EDIIIWNV-dS from the membranes, resulting in its retention within the cells.ConclusionsThis study presents an alternative approach for the recovery and partial purification of viral membrane proteins expressed in H. polymorpha. The factors influencing the effectiveness of this procedure and its potential use for the recovery of other integral membrane proteins are discussed.

Production and antigenicity analysis of a recombinant insulinoma associated protein-2 in HEK293 cells

Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

Identification of the immunogenic membrane proteins, Catalase, PgbA, and PgbB, as potential antigens against Helicobacter pylori.

This study aims to investigate the specific membrane antigens that are targeted by antibodies raised against H. pylori. Bovine milk antibodies were prepared using whole H. pylori, purified membrane proteins, or both. Enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments revealed that these immunogens triggered anti-H. pylori antibody production in milk. The highest antibody titer was induced by the mixture of whole bacteria and purified membrane proteins. The antibodies induced by mixed immunogens significantly inhibited H. pylori growth in vitro and were used to identify catalase, plasminogen-binding protein A (PgbA), and PgbB via western blotting, immunoprecipitation and two-dimensional western blotting (2D-WB) followed by LC-MS/MS. The immunogenicity of PgbA and PgbB was verified in mice vaccinated with their B-cell epitope vaccines. Following prophylactic vaccination of C57BL/6 mice, each of the three antigens alone and their combination reduced the weight loss in mice, increased antibody titers, and relieved the inflammatory status of the gastric mucosa following H. pylori infection. Catalase, PgbA, and PgbB could serve as valuable membrane antigens for the development of anti-H. pylori immunotherapies.

Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark

The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels. The production conditions were optimized and EfmRseP was purified by immobilized metal ion affinity chromatography followed by size-exclusion chromatography. The purification resulted in an overall yield of approximately 1 mg of pure protein per 3 g of wet-weight cell pellet. The structural integrity of the purified protein was confirmed using circular dichroism. We further assessed the expression and purification of RseP from E. faecium in the Gram-negative Escherichia coli. Detection of soluble protein failed in two of the three E. coli strains tested. Purification of EfmRseP expressed in E. coli C43(DE3) resulted in a protein with lower purity compared to EfmRseP expressed in L. plantarum. To our knowledge, this is the first time L. plantarum and the pSIP expression system have been applied for the production of membrane proteins.

Surfactant-Mediated Structural Modulations to Planar, Amphiphilic Multilamellar Stacks.

The hydrophobic effect, a ubiquitous process in biology, is a primary thermodynamic driver of amphiphilic self-assembly. It leads to the formation of unique morphologies including two highly important classes of lamellar and micellar mesophases. The interactions between these two types of structures and their involved components have garnered significant interest because of their importance in key biochemical technologies related to the isolation, purification, and reconstitution of membrane proteins. This work investigates the structural organization of mixtures of the lamellar-forming phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and two zwitterionic micelle-forming surfactants, being n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12 or DDAPS) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (O-Lyso-PC), when assembled by water vapor hydration with X-ray diffraction measurements, brightfield optical microscopy, wide-field fluorescence microscopy, and atomic force microscopy. The results reveal that multilamellar mesophases of these mixtures can be assembled across a wide range of POPC to surfactant (POPC:surfactant) concentration ratios, including ratios far surpassing the classical detergent-saturation limit of POPC bilayers without significant morphological disruptions to the lamellar motif. The mixed mesophases generally decreased in lamellar spacing (D) and headgroup-to-headgroup distance (Dhh) with a higher concentration of the doped surfactant, but trends in water layer thickness (Dw) between each bilayer in the stack are highly variable. Further structural characteristics including mesophase topography, bilayer thickness, and lamellar rupture force were revealed by atomic force microscopy (AFM), exhibiting homogeneous multilamellar stacks with no significant physical differences with changes in the surfactant concentration within the mesophases. Taken together, the outcomes present the assembly of unanticipated and highly unique mixed mesophases with varied structural trends from the involved surfactant and lipidic components. Modulations in their structural properties can be attributed to the surfactant's chemical specificity in relation to POPC, such as the headgroup hydration and the hydrophobic chain tail mismatch. Taken together, our results illustrate how specific chemical complexities of surfactant-lipid interactions can alter the morphologies of mixed mesophases and thereby alter the kinetic pathways by which surfactants dissolve lipid mesophases in bulk aqueous solutions.

A purified membrane protein from Akkermansia muciniphila blunted the sepsis-induced acute lung injury by modulation of gut microbiota in rats.

The gut microbiota has been implicated in the pathogenesis and progression of sepsis. Akkermansia muciniphila is considered to be a promising probiotic with reduced abundance in cecal ligation and puncture (CLP)-induced sepsis model, and its specific outer membrane protein (Amuc_1100) can partially recapitulate the probiotic function of Akkermansia muciniphila. However, its role in sepsis is unclear. This study aimed to investigate the effect of Amuc_1100 on the gut microbiota of septic rats, thereby improving the prognosis of septic acute lung injury (ALI). A total of 42 adult Sprague-Dawley (SD) rats were randomly divided into three groups: the sham control (SC group), the septic ALI induced by CLP method (CLP group), and administered Amuc_1100 by oral gavage (3µg/d) for 7 d before the CLP procedure (AMUC group). The survival of the three groups was recorded and the feces and lung tissues of rats were collected 24h after treatment for 16S rRNA sequencing and histopathological evaluation. Oral administration of Amuc_1100 improved the survival rate and alleviated lung histopathological damage induced by sepsis. Serum levels of pro-inflammatory cytokines and chemokines were substantially attenuated. Amuc_1100 significantly increased the abundance of some beneficial bacteria in septic rats. Additionally, the Firmicutes/Bacteroidetes ratio was low in septic rats, which was partially corrected by increasing Firmicutes and decreasing Bacteroidetes after oral administration of Amuc_1100 (p<0.05). In addition, Escherichia-Shigella, Bacteroides, and Parabacteroides were relatively enriched in septic rats, while in the AMUC group, their abundance was restored to levels similar to that of the healthy group. Amuc_1100 protects against sepsis by enhancing beneficial bacteria and reducing potential pathogenic bacteria. These findings indicate that Amuc_1100 can blunt CLP-induced ALI through the modulation of gut microbiota, thereby providing a new promising therapeutic target in sepsis.

Large-scale growth of C. elegans and isolation of membrane protein complexes.

Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and 'Protein S'), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.

Cloning, expression and purification of the outer membrane protein N from Gram-negative bacterial strains

To employ polyclonal antibodies against 6his-tags to detect the expression of recombinant EcOmpN in the E. coli BL21 (DE3) Omp8 Rosetta strain using a Western blot, the ompN gene segments from four Gram-negative bacterial strains (including E. coli of BL21 (DE3) Omp8 Rosetta, DH5α, Origami, and XL1-blue) were combined with the pET21b(+) vector to create recombinant plasmids, known as pET21b(+)/ompN-BOR, pET21b(+)/ompN-DHA, pET21b(+)/ompN-Ori, and pET21b(+)/ompN-XLB for production of four recombinant EcOmpN proteins, called BL21 (DE3) Omp8 Rosetta (BOR), DH5α (DHA), Origami (Ori) and XL1-blue (XLB), respectively. Each recombinant plasmid-carrying bacterial strain was induced with 0.5 mM IPTG for 4 h. Crude recombinant EcOmpN proteins were separated on a 12% SDS-PAGE and transferred to a nitrocellulose membrane and treated separately with anti-OmpN and anti-histidine tag polyclonal antibodies. E. coli BL21 (DE3) Omp8 Rosetta strains fully produced all four recombinant EcOmpN proteins as inclusion bodies. Following protein refolding procedures, the secondary structure of recombinant BOR protein was likewise shown in the stable trimeric form, whereas recombinant DHA, XLB and Ori proteins were demonstrated in monomeric form. The intrinsic fluorescence and substance interaction of recombinant BOR protein were further established and showed that this protein reacted with kanamycin more intensely than ampicillin.

Rationalizing the Optimization of Detergents for Membrane Protein Purification.

Membrane protein purification by means of detergents is key to isolating membrane-bound therapeutic targets. The role of the detergent structure in this process, however, is not well understood. Detergents are optimized empirically, leading to failed preparations, and thereby raising costs. Here we evaluate the utility of the hydrophilic-lipophilic balance (HLB) concept, which was introduced by Griffin in 1949, for guiding the optimization of the hydrophobic tail in first-generation, dendritic oligoglycerol detergents ([G1] OGDs). Our findings deliver qualitative HLB guidelines for rationalizing the optimization of detergents. Moreover, [G1] OGDs exhibit strongly delipidating properties, regardless of the structure of the hydrophobic tail, which delivers a methodological enabling step for investigating binding strengths of endogenous lipids and their role for membrane protein oligomerization. Our findings will facilitate the analysis of challenging drug targets in the future.

A generalizable protocol for expression and purification of membrane-bound bacterial phosphoglycosyl transferases in liponanoparticles

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community.

Overcoming problems to produce the recombinant protein LipL21 of Leptospira interrogans.

The production of leptospiral recombinant proteins in the soluble form and in high yield from Escherichia coli is still a challenge. This work presents the cloning, expression and purification of the outer membrane protein of Leptospira interrogans, LipL21, which isconsidered an interesting target for vaccine and diagnostics development. The expression profile and yield of LipL21 was compared after cloning in the vectors pAE, pET28a and pET-SUMO, and it wasobserved that LipL21 was expressed in a low amount with pAE vector. By using the pET-28a vector, protein expression was increased, but the majority of the product was obtained as inclusion bodies. As a highlight, using a pET-SUMO vector was shown to overcome the problems of low expression and solubility of the lipoprotein LipL21.

Delivery of nonbiologically-compatible membrane protein constructs to mammalian cells for functional characterization.

A hallmark of integral membrane protein biogenesis is the various cellular pathways that identify and tightly regulate protein fates. These quality control mechanisms identify misfolded from well-folded membrane proteins and target them for degradation or trafficking to the appropriate cellular membrane. A current limitation in membrane protein biophysics is that many engineered membrane proteins used in structural, molecular, and mechanistic studies; including mutations, truncations, and chimeras, do not satisfy cellular quality control requirements, limiting their ability for cellular characterization. Our recently developed membrane delivery method enables recombinantly expressed and purified membrane proteins to be delivered back to cells for functional and other characterization. Here we extend these methods to characterize the delivery and evaluate the function of a minimal TRPV1 ion channel by whole-cell patch-clamp electrophysiology and other techniques. This minimal construct is ∼35% of the full-length human TRPV1 receptor and does not satisfy cellular quality control, and is thereby excluded from trafficking to the plasma membrane. Delivery of minimal TRPV1 directly to the plasma membrane enables its characterization and shows that it retains polymodal activation by heat, protons, and capsaicin, analogously to the full-length TRPV1 protein. These outcomes are validated with other methods such as electron microscopy and NMR data, enabling a bridge between cellular and biophysical studies by bypassing cellular quality control and trafficking pathways.

Protein reconstitution into giant unilamellar vesicles from native nanodiscs.

The reconstitution of purified membrane proteins into lipid bilayers represents a tedious challenge as it relies mainly on empirically optimized purification protocols tailored to the target protein, including laborious detergent selection. The situation is even more complex when the protein's function or structural stability depends on its lipid environment. Using nanodisc-forming amphiphilic polymers helps circumnavigate these problems by allowing the extracted protein to stay in its native lipid environment. Yet, thus far, nanodiscs are of limited use for transport studies due to the problematic protein transfer to vesicles or planar bilayers. Here we exploited the recently introduced glycopolymer Glyco-DIBMA to purify integral membrane proteins such as the voltage-gated potassium ion channel KvAP, the voltage-gated sodium ion channel NavMs, the bacterial translocon complex SecYEG, the eukaryotic serotonin-gated cation channel 5Ht3A, as well as the phospholipid translocating scramblase TMEM16. We used fluorescence correlation spectroscopy to quantify the abundance of the labeled proteins in nanodiscs and optimize protein transfer into giant unilamellar vesicles (GUVs). We thus could achieve protein densities of roughly one copy per diffraction-limited focal area or ∼5400 copies in a GUV with a diameter of ∼20 µm. Such protein density is sufficient for visualizing the reconstituted proteins in vesicular membranes by conventional fluorescence microscopy. The proteins retain functionality, as our GUV influx and efflux studies demonstrated. This project was supported by a grant from the FWF (Austrian Science Fund): P34826.

Strategies for the Purification of Membrane Proteins.

Membrane proteins account for approximately 30% of the coding regions of all sequenced genomes, and they play crucial roles in many fundamental cell processes. However, there are relatively few membrane proteins with known three-dimensional structures. This is likely due to technical challenges associated with membrane protein extraction, solubilization, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non-covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, with the latter's extraction requiring phospholipid bilayer disruption using detergents or organic solvents. In this chapter, important considerations for membrane protein purification are addressed, with a focus on the initial stages of membrane protein solubilization, where problems are most frequently encountered. Protocols are outlined for the extraction of peripheral membrane proteins, solubilization of integral membrane proteins, and sample clean-up and concentration.

Fabrication of membrane proteins in the form of native cell membrane nanoparticles using novel membrane active polymers.

Membrane proteins are a widespread class of bio-macromolecules responsible for numerous vital biological processes and serve as therapeutic targets for a vast array of contemporary medications. For membrane protein isolation and purification, detergents have historically been used. Despite this, detergents frequently result in protein instability. Consequently, their application was limited. Recent detergent-free approaches have been invented. Among these, styrene-maleic acid lipid particle (SMALP), diisobutylene-maleic acid lipid particle (DIBMALP), and native cell membrane nanoparticle (NCMN) systems are the most prevalent. The NCMN system intends to create a library of membrane-active polymers suitable for high-resolution structure determination of membrane protein. Design, synthesis, characterization, and comparative application evaluations of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x, and NCMNP21b-x, are presented in this article. Although each NCMN polymer can solubilize distinct model membrane proteins and retain native lipids in NCMN particles, only the NCMNP21b-x family produces lipid-protein particles with ideal buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. NCMNP21b-x polymers that generate high-quality NCMN particles are particularly desirable for membrane protein structural biology.

Combining native mass spectrometry and lipidomics to uncover specific membrane protein-lipid interactions from natural lipid sources.

While it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative approach that combines native mass spectrometry (MS) and lipidomics to identify lipids retained by membrane proteins from natural lipid extracts. Our results reveal that the bacterial ammonia channel (AmtB) enriches specific cardiolipin (CDL) and phosphatidylethanolamine (PE) from natural headgroup extracts. When the two extracts are mixed, AmtB retains more species, wherein selectivity is tuned to bias headgroup selection. Using a series of natural headgroup extracts, we show TRAAK, a two-pore domain K+ channel (K2P), retains specific acyl chains that is independent of the headgroup. A brain polar lipid extract was then combined with the K2Ps, TRAAK and TREK2, to understand lipid specificity. More than a hundred lipids demonstrated affinity for each protein, and both channels were found to retain specific fatty acids and lysophospholipids known to stimulate channel activity, even after several column washes. Natural lipid extracts provide the unique opportunity to not only present natural lipid diversity to purified membrane proteins but also identify lipids that may be important for membrane protein structure and function.

Separating Inner and Outer Membranes of Escherichia coli by EDTA-free Sucrose Gradient Centrifugation.

The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.