Isolation Of Membrane Proteins Research Articles

Conformationally Restricted Monosaccharide-Cored Glycoside Amphiphiles: The Effect of Detergent Headgroup Variation on Membrane Protein Stability.

Detergents are widely used to isolate membrane proteins from lipid bilayers, but many proteins solubilized in conventional detergents are structurally unstable. Thus, there is major interest in the development of novel amphiphiles to facilitate membrane protein research. In this study, we have designed and synthesized novel amphiphiles with a rigid scyllo-inositol core, designated scyllo-inositol glycosides (SIGs). Varying the headgroup structure allowed the preparation of three sets of SIGs that were evaluated for their effects on membrane protein stability. When tested with a few model membrane proteins, representative SIGs conferred enhanced stability to the membrane proteins compared to a gold standard conventional detergent (DDM). Of the novel amphiphiles, a SIG designated STM-12 was most effective at preserving the stability of the multiple membrane proteins tested here. In addition, a comparative study of the three sets suggests that several factors, including micelle size and alkyl chain length, need to be considered in the development of novel detergents for membrane protein research. Thus, this study not only describes new detergent tools that are potentially useful for membrane protein structural study but also introduces plausible correlations between the chemical properties of detergents and membrane protein stabilization efficacy.

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.

Extraction and liposome reconstitution of membrane proteins with their native lipids without the use of detergents

Functional studies of membrane-bound channels, transporters or signal transducers require that the protein of interest resides in a membrane that separates two compartments. One approach that is commonly used to prepare these systems is to reconstitute the protein in liposomes. An intermediate step of this method is purification of the protein, which typically involves solubilization of the native membrane using detergent. The use of detergents often results in removal of lipids surrounding the protein, which may alter its structure and function. Here, we have employed a method for isolation of membrane proteins with a disc of their native lipids to develop an approach that allows transfer of the purified membrane protein to liposomes without the use of any detergents.

Membrane proteomics of cervical cancer cell lines reveal insights on the process of cervical carcinogenesis.

The available therapeutic approaches for cervical cancer can seriously affect the fertility potential of patient; thus, there is a pressing requirement for less toxic and targeted therapies. The membrane proteome is a potential source of therapeutic targets; however, despite the significance of membrane proteins in cancer, proteomic analysis has been a challenging task due to their unique biochemical properties. The aim of the present study was to develop an efficient membrane protein enrichment protocol, and to the best of our knowledge, to compare for the first time the expression pattern of membrane proteins of one normal cell line, HCK1T, and three cervical cancer cell lines, C33A, a human papilloma virus(HPV)-negative cell line, and two HPV-positive cell lines, SiHa (HPV16+) and HeLa (HPV18+). The study aimed to identify the proteins that are involved in cervical carcinogenesis and may constitute novel drug targets. Membrane protein isolation, liquid chromatography coupled with tandem mass spectrometry proteomics and bioinformatics analysis were performed in the membrane fraction of the informative cervical cell lines following a novel enrichment protocol. The percentages of membrane and transmembrane proteins in the enrichment protocol were higher compared with those of the corresponding data derived from total cell extract analysis. Differentially expressed proteins were detected by the comparison of the cervical cancer cell lines with the normal cell line. These proteins constitute molecular features of cancer pathology and participate in biological pathways relevant to malignancy, including 'HIPPO signaling', 'PI3K/Akt signaling', 'cell cycle: G2/M DNA damage checkpoint regulation' and 'EIF2 signaling'. These unique membrane protein identifications offer insights on a previously inaccessible region of the cervical cancer proteome, and may represent putative diagnostic and prognostic markers, and eventually therapeutic targets.

Molecular Mechanism of Lipid Nanodisk Formation by Styrene-Maleic Acid Copolymers

Experimental characterization of membrane proteins often requires solubilization. A recent approach is to use styrene-maleic acid (SMA) copolymers to isolate membrane proteins in nanometer-sized membrane disks, or so-called SMA lipid particles (SMALPs). The approach has the advantage of allowing direct extraction of proteins, keeping their native lipid environment. Despite the growing popularity of using SMALPs, the molecular mechanism behind the process remains poorly understood. Here, we unravel the molecular details of the nanodisk formation by using coarse-grained molecular dynamics simulations. We show how SMA copolymers bind to the lipid bilayer interface, driven by the hydrophobic effect. Due to the concerted action of multiple adsorbed copolymers, large membrane defects appear, including small, water-filled pores. The copolymers can stabilize the rim of these pores, leading to pore growth and membrane disruption. Although complete solubilization is not seen on the timescale of our simulations, self-assembly experiments show that small nanodisks are the thermodynamically preferred end state. Our findings shed light on the mechanism of SMALP formation and on their molecular structure. This can be an important step toward the design of optimized extraction tools for membrane protein research.

Hydrolysis Activity of Pyloric Cecal Enterocytes of Brown Trout (Salmo trutta) toward Monoacylglycerol and Lysophosphatidylcholine.

Some lipid digestion pathways in fish deviate from those in mammals, and many differences may also be species dependent. This report describes a pathway for monoacylglycerol (MAG) and lysophospholipid absorption by intestinal enterocytes in brown trout that may be of significance in salmonids. When culturing primary cells in a medium containing 1- and 2-MAG, we observed a massive hydrolysis of unesterified fatty acids. The hydrolysis activity was retained in the medium even after the removal of the cells. To further characterize these activities, both extracellular and isolated membrane proteins were tested for lipase activity toward triacylglycerol (TAG), diacylglycerol (DAG), MAG, phosphatidylcholine (PtdCho), and lysoPtdCho. In both cases, the main hydrolyzing activity was toward MAG followed by lysoPtdCho with very little activity toward DAG, TAG, or PtdCho. The extracellular and membrane proteins were partially purified by fast protein liquid chromatography and identified by proteomics (liquid chromatography-tandem mass spectrometry) focusing on lipase/hydrolase enzymes. In the membrane protein fraction, the data suggested that MAG was produced as an intermediate in the hydrolysis of lysoPtdCho by either lysophospholipase C or lysophospholipase D activity. Both abhydrolase-domain-containing protein 6 and abhydrolase-domain-containing protein 12 were identified in the membrane protein and they could be responsible for the hydrolysis of MAG. In the culture medium, low-peptide matches were found for ABHD6 and phospholipases and further studies are needed. In summary, trout enterocytes are capable of hydrolyzing MAG and lysoPtdCho. The enzymes are both extracellular and membrane bound. The pathways may be of significance during lipid absorption in fish lacking a 1,3 specific pancreatic lipase.

Investigation of Thermodynamic Dissociation Kinetics to Determine the Binding Strengths within a Membrane Protein Complex: Photosystem II Supercomplex

Protein-protein interactions as well as the formations of protein complexes have been intensively studied by various methods in both in vivo and in vitro. However, although membrane proteins have important roles in cells, interactions within membrane protein complexes have been hardly studied than that within soluble proteins due to the lack of suitable methods, though one approach using mass spectrometry has been recently developed to elucidate membrane protein complexes. Here, we present an approach to determine binding strengths within a membrane protein complex by investigating the thermodynamic dissociation kinetics of an isolated membrane protein complex using time-resolved fluorescence spectroscopy. We demonstrated different bindings of light-harvesting complex II (LHCII) trimers in a photosystem II (PSII) supercomplex isolated from a green alga Chlamydomonas reinhardtii, where the different binding types of the LHCII trimers have been indirectly predicted by the presence or absence of LHCII trimers in negatively stained PSII supercomplex particles observed by electron microscopy but have not been determined. As a result of analysis on thermodynamic dissociation kinetics, we identified three different activation energies for the dissociations of LHCII trimers, which imply the presence of multiple hydrogen bonds and/or ion-dipole bonds between LHCII trimers and core complexes.

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.

Mini-Scale Isolation and Preparation of Plasma Membrane Proteins from Potato Roots for LC/MS Analysis.

Plasma membrane (PM) proteins are of special interest due to their function in exchanging material and information with the external environment as well as their role in cellular regulation. In quantitative proteomic studies PM proteins are underrepresented mostly because they constitute only small percent of all membrane proteins. Strong demand is placed on plasma membrane enrichment methods. For decades two-phase partitioning Dextran T500/PEG 3350 isolation protocols were applied for many different animal and plant species and also a variety of tissue types. The typical quantity of material used in the enrichment protocols is 10-30g of fresh weight. The main difficulty of working with in vitro cultivated plants is the low amount of material, especially when roots are examined. In addition, roots are frequently characterized by low protein concentrations. Our protocol established for roots of in vitro cultivated potato plants is adjusted to amounts of fresh weight not exceeding 7.5g and allows studying the plasma membrane proteome by LC-MS.

Controlling Styrene Maleic Acid Lipid Particles through RAFT.

The ability of styrene maleic acid copolymers to dissolve lipid membranes into nanosized lipid particles is a facile method of obtaining membrane proteins in solubilized lipid discs while conserving part of their native lipid environment. While the currently used copolymers can readily extract membrane proteins in native nanodiscs, their highly disperse composition is likely to influence the dispersity of the discs as well as the extraction efficiency. In this study, reversible addition-fragmentation chain transfer was used to control the polymer architecture and dispersity of molecular weights with a high-precision. Based on Monte Carlo simulations of the polymerizations, the monomer composition was predicted and allowed a structure-function analysis of the polymer architecture, in relation to their ability to assemble into lipid nanoparticles. We show that a higher degree of control of the polymer architecture generates more homogeneous samples. We hypothesize that low dispersity copolymers, with control of polymer architecture are an ideal framework for the rational design of polymers for customized isolation and characterization of integral membrane proteins in native lipid bilayer systems.

Solubilization of human cells by the styrene–maleic acid copolymer: Insights from fluorescence microscopy

Extracting membrane proteins from biological membranes by styrene–maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes.

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices.

The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. These methods should be applicable to a wide range of membrane proteins.

Screening for the best detergent for the isolation of placental membrane proteins

Although membrane proteins (MPs) play crucial roles in physiological processes, information on them are insufficient, mostly due to their peculiar nature and surrounding which demand specific procedures for their extraction (using detergents) and analysis. A pallet of ten detergents and β-cyclodextrin was employed to investigate their efficiency in extracting total placental MPs, glycoproteins and insulin-like growth factor receptors (IR/IGF1R/IGF2R). Regardless of detergent used, the identity of major extracted proteins was the same. Glycoproteins extracted with Triton X-100 contained the greatest variety and quantity of glycans recognised by fifteen lectins, pointing to this detergent as universal medium for the extraction of membrane glycoproteins. Glycoproteins extracted using Brij 35 exhibited weak interaction with only seven lectins and were differently recognised by lectins of the similar glycan specificity. Brij 35, Tween 20, saponin and digitonin selectively extracted IGF2R compared to other two receptors. Pilot experiments should be conducted in order to choose adequate detergent for the extraction of specific MP. To obtain preparations enriched in specific receptor of the insulin/IGF system sequential solubilisation of placental MPs can be proposed: to use Brij 35 to extract IGF2R and subject the insoluble remaining suspension to Triton X-114 in order to extract most of IGF1R with small amounts of IR.

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

Styrene-Maleic acid (SMA) copolymers have emerged as a powerful alternative to detergents for applications in membrane research. Most notably, these amphipathic polymers can be used to directly extract and purify membrane proteins from intact cells of different organisms in the form of ‘‘native nanodiscs’’. These particles stabilize the protein in a near native environment comprising conserved native lipids as well as other membrane components that allow for the study of preferential interactions by biochemical analysis.To evaluate the general applicability of SMA-mediated membrane protein extraction from biomembranes, we employed fluorescence microscopy to visualize SMA solubilization of HeLa cells in real time. Our results indicate that SMA solubilization of (human) cells is a multi-step process that is not specific for any (sub)cellular membrane. However, we found solubilization kinetics of plasma membrane-localized proteins to vary markedly for different proteins, suggesting a strong influence of membrane organization and domain formation.To assess the influence of membrane properties on solubilization preferences of SMA in more detail, we employed an approach of partial solubilization of model membranes of different compositions. In lipid mixtures that form homogeneous bilayers, we found that SMA exhibits a high degree of promiscuity with respect to lipid headgroups. However, in phase-separating systems of fluid phases with either gel-phase or liquid-ordered phases, lipids in the fluid phase were solubilized with a strong preference.Our findings support the use of SMA for the study of lipid-protein interactions of membrane proteins and suggest applications for the isolation of membrane proteins from ordered domains in phase-separated membranes.

Optimizing Conditions for the Isolation of Membrane Proteins by Styrene-Maleic Acid Copolymers: A Case Study on E. Coli Inner Membranes

Styrene-Maleic acid (SMA) copolymers have emerged as a powerful alternative to detergents for the extraction of membrane proteins from cellular membranes. This approach has been successfully demonstrated for proteins present in the membranes of a variety of organisms including all major systems used for heterologous expression. However, for downstream characterization often high amounts of proteins are required, which remains a challenge in some cases. Extraction of proteins from the membrane and purification of the solubilized material are two major bottlenecks in this process.Here, we report a systematic approach to optimize conditions for the use of SMA to extract the tetrameric potassium channel KcsA from the inner membrane of Escherichia coli, by varying different parameters such as SMA-to-membrane ratio, ionic strength, pH, polymer type and polymer length. Furthermore, conditions for the purification of the channel via Ni-affinity of its poly-His tag have been optimized. Implications of our findings will be discussed in the general context of membrane protein solubilization by SMA.

Homology Modeling of Human Concentrative Nucleoside Transporters (hCNTs) and Validation by Virtual Screening and Experimental Testing to Identify Novel hCNT1 Inhibitors.

The nucleoside transporter family is an emerging target for cancer, viral and cardiovascular diseases. Due to the difficulty in the expression, isolation and crystallization of membrane proteins, there is a lack of structural information on any of the mammalian and for that matter the human proteins. Thus the objective of this study was to build homology models for the three cloned concentrative nucleoside transporters hCNT1, hCNT2 and hCNT3 and validate them for screening towards the discovery of much needed inhibitors and probes. The recently reported crystal structure of the Vibrio cholerae concentrative nucleoside transporter (vcCNT), has satisfactory similarity to the human CNT orthologues and was thus used as a template to build homology models of all three hCNTs. The Schrödinger modeling suite was used for the exercise. External validation of the homology models was carried out by docking a set of recently reported known hCNT1 nucleoside class inhibitors at the putative binding site using induced fit docking (IDF) methodology with the Glide docking program. Then, the hCNT1 homology model was subsequently used to conduct a virtual screening of a 360,000 compound library, and 172 compounds were obtained and biologically evaluated for hCNT 1, 2 and 3 inhibitory potency and selectivity. Good quality homology models were obtained for all three hCNTs as indicated by interrogation for various structural parameters and also external validated by docking of known inhibitors. The IDF docking results showed good correlations between IDF scores and inhibitory activities; particularly for hCNT1. From the top 0.1% of compounds ranked by virtual screening with the hCNT1 homology model, 172 compounds selected and tested for against hCNT1, hCNT2 and hCNT3, yielded 14 new inhibitors (hits) of (i.e., 8% success rate). The most active compound exhibited an IC50 of 9.05 μM, which shows a greater than 25-fold higher potency than phlorizin the standard CNT inhibitor (IC50 of 250 μM). We successfully undertook homology modeling and validation for all human concentrative nucleoside transporters (hCNT 1, 2 and 3). The proof-of-concept that these models are promising for virtual screening to identify potent and selective inhibitors was also obtained using the hCNT1 model. Thus we identified a novel potent hCNT1 inhibitor that is more potent and more selective than the standard inhibitor phlorizin. The other hCNT1 hits also mostly exhibited selectivity. These homology models should be useful for virtual screening to identify novel hCNT inhibitors, as well as for optimization of hCNT inhibitors.

Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells.

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

Reconstitution of Mitochondrial Membrane Proteins into Nanodiscs by Cell-Free Expression.

The isolation and characterization of mitochondrial membrane proteins is technically challenging because they natively reside within the specialized environment of the lipid bilayer, an environment that must be recapitulated to some degree during reconstitution to ensure proper folding, stability, and function. Here we describe protocols for the assembly of a membrane protein into lipid bilayer nanodiscs in a series of cell-free reactions. Cell-free expression of membrane proteins circumvents problems attendant with in vivo expression such as cytotoxicity, low expression levels, and the formation of inclusion bodies. Nanodiscs are artificial membrane systems comprised of discoidal lipid bilayer particles bound by annuli of amphipathic scaffold protein that shield lipid acyl chains from water. They are therefore excellent platforms for membrane protein reconstitution and downstream solution-based biochemical and biophysical analysis. This chapter details the procedures for the reconstitution of a mitochondrial membrane protein into nanodiscs using two different types of approaches: cotranslational and posttranslational assembly. These strategies are broadly applicable for different mitochondrial membrane proteins. They are also applicable for the use of nanodiscs with distinct lipid compositions that are biomimetic for different mitochondrial membranes and that recapitulate lipid profiles associated with pathological disorders in lipid metabolism.

Isolation and characterization of outer membrane proteins (OMPs) from Salmonella Gallinarum in chicken and antibiogram of the isolates

Salmonella isolates should be distinguished as it may assist in tracing the source of an outbreak and monitoring trends in antimicrobial resistance associated with a particular type. The specific detection of these Salmonella serotypes is therefore extremely important in order to attribute an isolate to a previously known epidemic outbreak. The present investigation was to isolate and identify S. Gallinarum, to study variation in the profile of outer membrane proteins (OMPs) and to determine in vitro antibiogram of S. Gallinarum in poultry. A total of 228 faecal samples and 22 visceral samples suspected for Salmonellosis were collected, of these 15 samples (6.0%) were found positive for S. Gallinarum. In the present study, rfbS gene sequence was helpful in the serotype-specific detection of S. Gallinarum giving a 187 bp product. Salmonella Gallinarum crude protein extracts determined by SDSPAGE showed migration of OMPs as several bands at approximate moleculer weights of appx. 45 kDa, 55 kDa, 64 kDa, 65 kDa, 74 kDa, 110 kDa, 120 kDa, 135 kDa, 150 kDa,155 kDa, 200 kDa and above 200 kDa. The study indicated a definite variation in the profile of OMPs of various Salmonella Gallinarum strains with major OMPs in the range of appx 80-100 kDa which could be the target for vaccine production. All the isolates tested against 14 antimicrobial agents showed variable susceptibility pattern with highest resistance to nalidixic acid, ampicillin and sulphadiazine and sensitivity to chloramphenicol, gentamicin and enrofloxacin.

Casein precipitation by acid and rennet coagulation of buttermilk: Impact of pH and temperature on the isolation of milk fat globule membrane proteins

Milk fat globule membrane (MFGM) proteins make up only 2–3% of the total protein content in buttermilk. Prior isolation of buttermilk MFGM material is therefore required for further investigation of its functional properties and application in food systems. Rennet induced coagulation has been applied to remove caseins, but 30–70% of MFGM proteins are entrapped in the casein curd under classical cheese making conditions. Therefore, factors influencing the rennet as well as acid induced coagulation of buttermilk with regard to a reduction of MFGM protein losses into the casein curd, i.e., pH, temperature and pre-heat treatment of buttermilk, were examined. Complete casein removal from buttermilk was reached using both coagulation methods, whereas retention of MFGM proteins in the serum phase was higher during renneting. Rennet induced coagulation at higher pH and lower temperatures resulted in weaker gel structures, which increased the yield of MFGM proteins in the buttermilk whey.