Isolation Of Integral Membrane Proteins Research Articles

Strategies for successful isolation of a eukaryotic transporter

The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643–672 (OsBOR3Δ1-642). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-β-d-maltoside (DDM). The OsBOR3Δ1-642 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3Δ1-642 than DDM. Isolation of OsBOR3Δ1-642 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3Δ1-642 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3Δ1-642 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles.

Analysis of styrene maleic acid alternating copolymer supramolecular assemblies in solution by small angle X-ray scattering

Over the past decade, Styrene Maleic Acid alternating copolymers (SMA) have gained interest as an alternative to detergent solubilization for the isolation of integral membrane proteins. The formation of SMA lipid particles (SMALPs) presents a novel opportunity to isolate the proximal membrane environment, encompassing and throughout membrane proteins in vitro. Neither the organization or structure of SMAs in an aqueous buffer nor the mechanism by which SMA transforms the membrane bilayer into a SMALP is known. This study investigates the shape and size of diverse SMA polymer complexes/aggregates in solution at various pH, ionic strength, SMA concentration and temperature, analyzed by small angle X-ray scattering. It is clear that SMAs of differing physicochemical properties (styrene to maleic acid ratio, length of copolymer fragments and functionalization) display highly variable sizes/shapes in solution over a range of environmental conditions. The SMA supramolecular aggregates exhibit similar prolate ellipsoidal geometry of varying size, dependent on the degree of hydrophobicity of the SMA copolymer. At elevated temperature, particles composed of SMAs enriched in styrene increase in both radius of gyration and maximum particle diameter. Interestingly, we observe a correlation between the SMALP dimensions and that of native membranes. Future work will investigate if there may be a complimentary relationship between SMA aggregate dimensions and bilayer thickness (and/or protein transmembrane domain thickness), similar to what has been observed for tandem facial amphiphiles.

Enrichment of integral membrane proteins from small amounts of brain tissue

Subcellular fractionation represents an essential technique for functional proteome analysis. Recently, we provided a subcellular fractionation protocol for minute amounts of tissue that yielded a nuclear fraction, a membrane and organelle fraction, and a cytosolic fraction. In the current study, we attempted to improve the protocol for the isolation of integral membrane proteins, as these are particularly important for brain function. In the membrane and organelle fraction, we increased the yield of membranes and organelles by about 50% by introducing a single re-extraction step. We then tested two protocols towards their capacity to enrich membrane proteins present in the membrane and organelle fraction. One protocol is based on sequential solubilization using subsequent increases of chaotropic conditions such as urea, thereby partitioning hydrophobic proteins from hydrophilic ones. The alternative protocol applies high-salt and high-pH washes to remove non-membrane proteins. The enrichment of membrane proteins by these procedures, as compared to the original membrane and organelle fraction, was evaluated by 16-BAC-SDS-PAGE followed by mass spectrometry of randomly selected spots. In the original membrane and organelle fraction, 7 of 50 (14%) identified proteins represented integral membrane proteins, and 15 (30%) were peripheral membrane proteins. In the urea-soluble fraction, 4 of 33 (12%) identified proteins represented integral membrane proteins, and 10 (30%) were peripheral membrane proteins. In the high-salt/high-pH resistant sediment, 12 of 45 (27%) identified proteins were integral membrane proteins and 13 (29%) represented peripheral membrane proteins. During the analysis, several proteins involved in neuroexocytosis were detected, including syntaxin, NSF, and Rab3-interaction protein 2. Taken together, differential centrifugation in combination with high-salt and high-pH washes resulted in the highest enrichment of integral membrane proteins and, therefore, represents an adequate technique for region-specific profiling of membrane proteins in the brain.

Rapid Purification and Mass Spectrometric Characterization of Mitochondrial NADH Dehydrogenase (Complex I) from Rodent Brain and a Dopaminergic Neuronal Cell Line

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research

Detergents are crucial to the isolation of integral membrane proteins. During membrane protein purification, it is useful to accurately quantify detergent, especially if concentration steps have been used. Previously, this has been difficult and time-consuming. We present a simple, rapid, and sensitive procedure for the quantification of glycosidic and bile salt-based detergents such as dodecylmaltoside, octylglucoside, and CHAPS. The method directly quantifies sugar or cholate moieties via colorimetric reactions with phenol and sulfuric acid. A number of detergents have been screened, and the assay has been validated in the presence of commonly used reagents. In addition to determining the overall detergent concentration in solution, the procedure allows accurate quantification of specific binding of glycosidic or bile salt-based detergents to purified membrane proteins. Both the colorimetric method and the radiometric 14C method were used to determine detergent binding to two integral membrane proteins: the cytochrome cbb 3 oxidase from Pseudomonas stutzeri and the turkey β-adrenergic receptor. Both methods gave similar results. After separating monomeric glycosidic detergent from micellar solutions by ultrafiltration, we used the colorimetric method to determine the concentration of monomeric detergent present. We observed that values obtained are in close agreement with previously determined critical micelle concentrations.

Isolation of integral membrane proteins of Leishmania promastigotes and evaluation of their prophylactic potential in hamsters against experimental visceral leishmaniasis

The integral membrane proteins (IMP's) of promastigotes of two virulent strain of Leishmania (L.) donovani Dd8 and Leishmania (L.) infantum LV9 and one avirulent viscerotropic strain Leishmania tropica UR6 were extracted by phase separation technique using a non-ionic detergent “Triton X-114”. This detergent is homogeneous at 0 °C but divides in an aqueous phase and a detergent phase at above 20 °C. The phase partition resulted in solubilisation of hydrophilic proteins in aqueous phase, and IMP's with an amphiphilic nature were recovered in the detergent phase. The strain wise quantitative recovery rates of IMP's were estimated. These proteins were purified by chill methanol centrifugation and used as vaccinogen, alone or in combination with adjuvant against L. donovani challenge in hamster model. Among all the combinations, hamsters immunised with IMP of L. donovani (Dd8) in combination with CFA resulted in 75% parasite inhibition in spleen ( P < 0.001), however, 61.14% ( P < 0.001) and 77.60% ( P < 0.001) parasitic inhibition was achieved in liver and bone marrow respectively as compared to their unvaccinated counter part. Similar combinations with UR6 and LV9 strain inhibited the parasite establishment up to 65.12% ( P < 0.001) and 66.87% ( P < 0.001), respectively on splenic site. The specific IgG level against (Dd8 strain) soluble leishmania promastigote antigen was monitored at different stages by enzyme linked immunosorbent assay (ELISA) corresponds to the level of parasitic establishment. Similar observations were made in cases of LV9 and UR6 strains. However, significant lymphoproliferative response to IMPs of Dd8 strain (SI 3.5–4.9, P < 0.001) was noticed in all IMP + CFA vaccinated animals. Thus, this study will provide a lead for more manipulative trials to develop a subunit vaccine against the fatal disease.

Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification.

Acrylamide concentration, urea content, and the trailing ion used for sodium dodecyl sulfate (SDS)-gels modify electrophoretic protein mobilities in a protein-dependent way. Varying these parameters we coupled two SDS-gels to a two-dimensional (2-D) electrophoresis system. Protein spots in 2-D gels are dispersed around a diagonal. Hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the novel technique. Mass spectrometric identification of previously unaccessible hydrophobic proteins is now possible.

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

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.

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: Application to the purification of the fusion protein of the human immunodeficiency virus type 1

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.

Isolation of an integral membrane glycoprotein by chloroform-methanol extraction and C 3-reversed-phase high-performance liquid chromatography

Methodology is presented for the isolation of integral membrane proteins and applied to the purification of the major myelin glycoprotein, P 0. This isolation scheme depends on the detergent solubilization of an isoosmotically extracted membrane fraction from sciatic nerve endoneurium, followed by the removal of lipids and detergent by chloroform/methanol extraction. The resulting membrane proteins are readily dissolved in acetic acid/water ( 1 1 ) and directly analyzed by reversed-phase high-performance liquid chromatography. The hydrophobic nature of the intrinsic membrane protein mixture results in strong binding to a C 8 stationary phase, leading to poor resolution and yields. These problems can be eliminated by employing a C 3 alkylsilane column, thereby allowing separation of the protein components and the isolation of P 0. The purified P 0 has an amino-terminal sequence that matches that predicted from nucleotide sequencing, and the glycoprotein contains the expected amount of sialic acid. This latter finding indicates that the isolation procedure is not detrimental to the complex-type oligosaccharide structure of P 0 and should make the methodology readily applicable to the purification of other integral membrane proteins and glycoproteins.