Purification and characterisation of two cancer cachexia factors

Cachexia is a wasting phenomenon that often accompanies malignant disease. Its manifestation is associated with shortened survival and reduced responsiveness to anti-tumour therapy and as yet there is no established, effective amelioratory treatment. The MAC 16 model of cancer cachexia has been shown by many studies to closely mirror the human condition. Thus, cachexia is mediated by the presence of a small, slow-growing solid tumour that is mainly resistant to chemotherapy. In addition, the condition is largely attributable to aberrations in metabolic processes, while weight loss due to anorexia is negligible. Cachexia induced by the MAC 16 tumour, has been shown to be mediated by the production of tumour-derived circulatory catabolic factors, and the further elucidation of the structure of these molecules contributes towards the main content of this report. Thus, a factor with in vitro lipid-mobilising activity has been purified from the MAC 16 tumour, and has been found to have similarities to tumour-derived lipolytic factors published to date. Further work demonstrated that this factor was also purifiable from the urine of a patient with pancreatic cancer, and that it was capable of inducing weight loss in non tumour-bearing mice. Sequence analysis of the homogeneous material revealed an identity to Zn-α-2-glycoprotein, the significance of which is discussed. An additional factor, first detected as a result of its specific reactivity with a monoclonal antibody produced by fusion of splenocytes from MAC 16 tumour-bearing mice with mouse BALB/c myeloma cells, was identified as a co-purificant during studies to isolate the lipolytic factor. Subsequent purification of this material to homogeneity resulted in the determination of 18 of the N-terminal amino acids and revealed the highly glycosylated nature of its structure. Thus, this material (P24) was found to have an apparent molecular mass of 24kD of which 2kD was due to protein, while the remainder (92%) was due to the presence of carbohydrate groups. Sequence analysis of the protein core of P24 revealed an identity with Streptococcal pre-absorbing antigen (PA-Ag) in 11 of the amino acids, and the significance of this is discussed. P24 was shown to induce muscle protein breakdown in vitro and to induce cachexia in vivo, as measured by the depletion of fat (29%) and muscle (14%) tissue in the absence of a reduction of food and water intake. Further studies revealed that the same material was purifiable from the urine of patients with pancreatic cancer and was found to be detectable in the urine of cancer patients with weight loss greater than l.Skg/month. Thus, cachexia induced by the MAC 16 tumour in mice and by malignant disease in humans may be induced by similar mediators. Attempts to isolate the gene for P24 using information provided by the N-terminal protein sequence were unsuccessful. This was probably due to the low abundance o[ the material, as determined by protein purification studies; and the nature of the amino acids of the N-terminal sequence, which conferred a high degree o[ degeneracy to the oligonucleotides designed for the polymerase chain reaction.

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

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

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.

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required. © 2007 Wiley Periodicals, Inc.

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Detergent-free purification of ABC (ATP-binding-cassette) transporters

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up awide range of possibilities for the future study of their structure and function. © The Authors Journal compilation © 2014 Biochemical Society.

G-protein-coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

Efficient purification of chromatin architectural proteins:histones, HMGB proteins and FKBP3 (FKBP25) immunophilin

A two-step process of high ionic strength lysis of chicken erythrocyte cell nuclei followed by cation-exchange chromatography has separated at very high yield all the histone and HMGB (high-mobility group B) nuclear proteins, except the less-soluble histone tetramers. Surprisingly high yields of the nuclear immunophilin FKBP3 (FKBP25) and Hsp70 (heat-shock protein 70) co-fractionate with HMGB1 and HMGB3. Furthermore, these proteins can be separated by anion-exchange chromatography. The purified nuclear proteins retain their native, post-translational modification (PTM) marks, including those associated with chromatin-fibre remodelling. These marks are intimately associated with the control of the cell cycle. The methods herein are therefore of value for targeting these and other nuclear proteins for future proteomic studies in healthy and diseased cells. This journal is © 2012 The Royal Society of Chemistry.

Methyl chloride purification for the silicone industry

This study experimentally investigated methyl chloride (MeCl) purification method using an inhouse designed and built volumetric adsorption/desorption rig. MeCl is an essential raw material in the manufacture of silicone however all technical grades of MeCl contain concentrations (0.2 - 1.0 % wt) of dimethyl ether (DME) which poison the process. The project industrial partner had previously exhausted numerous separation methods, which all have been deemed not suitable for various reasons. Therefore, adsorption/desorption separation was proposed in this study as a potential solution with less economic and environmental impact. Pure component adsorption/desorption was carried out for DME and MeCl on six different adsorbents namely: zeolite molecular sieves (types 4 Å and 5 Å); silica gels (35-70 mesh, amorphous precipitated, and 35-60 mesh) and granular activated carbon (type 8-12 mesh). Subsequent binary gas mixture adsorption in batch and continuous mode was carried out on both zeolites and all three silica gels following thermal pre-treatment in vacuum. The adsorbents were tested as received and after being subjected to different thermal and vacuum pre-treatment conditions. The various adsorption studies were carried out at low pressure and temperature ranges of 0.5 - 3.5 atm and 20 - 100 °C. All adsorbents were characterised using Brunauer Emmett Teller (BET), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDXA) to investigate their physical and chemical properties. The well-known helium (He) expansion method was used to determine the empty manifold and adsorption cell (AC) regions and respective void volumes for the different adsorbents. The amounts adsorbed were determined using Ideal gas laws via the differential pressure method. The heat of adsorption for the various adsorbate-adsorbent (A-S) interactions was calculated using a new calorimetric method based on direct temperature measurements inside the AC. Further adsorption analysis included use of various empirical and kinetic models to determine and understand the behaviour of the respective interactions. The gas purification behaviour was investigated using gas chromatography and mass spectroscopy (GC-MC) analysis. Binary gas mixture samples were syringed from the manifold iii and AC outlet before and after adsorption/desorption analysis through manual sample injections into the GC-MS to detect and quantify the presence of DME and ultimately observe for methyl chloride purification. Convincing gas purification behaviour was confirmed using two different GC columns, thus giving more confidence on the measurement reliability. From the single pure component adsorption of DME and MeCl on the as received zeolite 4A subjected to 1 h vacuum pre-treatment, both gases exhibited pseudo second order adsorption kinetics with DME exhibiting a rate constant nearly double that of MeCl thus suggesting a faster rate of adsorption. From the adsorption isotherm classification both DME and MeCl exhibited Type II and I adsorption isotherm classifications, respectively. The strength of bonding was confirmed by the differential heat of adsorption measurement, which was found to be 23.30 and 10.21 kJ mol-1 for DME and MeCl, respectively. The former is believed to adsorb heterogeneously through hydrogen bonding whilst MeCl adsorbs homogenously via van der Waal’s (VDW) forces. Single pure component adsorption on as received zeolite 5A, silica gels (35-70, amorphous precipitated and 35-60) resulted in similar adsorption/desorption behaviour in similar quantities (mol kg-1). The adsorption isotherms for DME and MeCl on zeolite 5A, silica gels (35-70, amorphous precipitated and 35-60) and activated carbon 8-12 exhibited Type I classifications, respectively. Experiments on zeolite 5A indicated that DME adsorbed stronger, faster and with a slightly stronger strength of interaction than MeCl but in lesser quantities. On the silica gels adsorbents, DME exhibited a slightly greater adsorption capacity whilst adsorbing at a similar rate and strength of interaction compared to MeCl. On the activated carbon adsorbent, MeCl exhibited the greater adsorption capacity at a faster rate but with similar heats of adsorption. The effect of prolonged vacuum (15 h), thermal pre-treatment (150 °C) and extended equilibrium time (15 min) were investigated for the adsorption behaviour of DME and MeCl on both zeolites 4A and 5A, respectively. Compared to adsorption on as received adsorbents subjected to 1 h vacuum the adsorption capacities for DME and MeCl were found to increase by 1.95 % and 20.37 % on zeolite 4A and by 4.52 % and 6.69 % on zeolite 5A, respectively. In addition the empirical and kinetic models and differential heats of adsorption resulted in more definitive fitting curves and trends due to the true equilibrium position of the adsorbate with the adsorbent. Batch binary mixture adsorption on thermally and vacuum pre-treated zeolite 4A demonstrated purification behaviour of all adsorbents used for MeCl streams containing DME impurities, with a concentration as low as 0.66 vol. %. The GC-MS analysis showed no DME detection for the tested concentration mixtures at the AC outlet after 15 or 30 min, whereas MeCl was detectable in measurable amounts. Similar behaviour was also observed when carrying out adsorption in continuous mode. On the other hand, similar studies on the other adsorbents did not show such favourable MeCl purification behaviour. Overall this study investigated a wide range of adsorbents (zeolites, silica gels and activated carbon) and demonstrated for the first time potential to purify MeCl streams containing DME impurities using adsorption/desorption separation under different adsorbent pre-treatment and adsorption operating conditions. The study also revealed for the first time the adsorption isotherms, empirical and kinetic models and heats of adsorption for the respective adsorbentsurface (A-S) interactions. In conclusion, this study has shown strong evidence to propose zeolite 4A for adsorptive purification of MeCl. It is believed that with a technical grade MeCl stream competitive yet simultaneous co-adsorption of DME and MeCl occurs with evidence of molecular sieiving effects whereby the larger DME molecules are unable to penetrate through the adsorbent bed whereas the smaller MeCl molecules diffuse through resulting in a purified MeCl stream at the AC outlet. Ultimately, further studies are recommended for increased adsorption capacities by considering wider operating conditions, e.g. different adsorbent thermal and vacuum pre-treatment and adsorbing at temperatures closer to the boiling point of the gases and different conditions of pressure and temperature.

Detergent-free membrane protein purification

Membrane proteins are localised within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilising or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilising protein structure is time consuming and expensive. Here we present a recently-developed method using a styrene maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.

Novel Purification of Archaeal Rnase P Protein, Pfu Rpp38, for Nmr Studies

Dr. Kenneth Murray, Ph.D. Assistant Professor of Biology Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving 5' leader sequences during tRNA maturation. RNase P comprises one essential RNA, and one protein subunit in eubacteria, five proteins in archaea, and ten in humans. Due to its homology to human RNase P, its higher stability, and simpler structure; extensive studies have been conducted utilizing the enzyme from the archaeal hyperthermophile, Pyrococcus furious (Pfu). Previous studies identified only four protein subunits associated with the archaeal RNase P. This fourprotein reconstituted particle, however, had an optimal temperature of 55°C, compared to the optimal 70°C of the wild type RNase P. Additional probing of the organism's genome database revealed a fifth RNase P protein subunit, RPP38. To facilitate further investigations of Pfu RNase complexes, we sought to develop a protocol for the purification ofRPP38. Our results, presented herein, represent the first known expression.purification protocol developed for RPP38. Briefly, we synthesized an N-terminal6x-His RPP38 fusion construct, reengineered to contain a Tobacco Etch Virus (TEV) protease cleavage site. Purification was achieved via immobilized metal affinity chromatography and reversed phase high performance liquid chromatography. Following purification the 6X-His affinity tag was removed via TEV cleavage, thus regenerating the native RPP38 protein. Purity and identity of RPP38 were confirmed by sodium dodecylsulfate - polyacrylamide gel electrophoresis and mass spectrometry, respectively. Our work is expected to contribute to our understanding ofRNase P function and tRNA maturation by providing an efficient, facile technique to express and purify Pfu RNase protein RPP38 as a means to facilitate structural and functional analyses.

Purification et identification d'une protéine de la membrane plasmique endothéliale : molécules d'adhésion de la microvascularisation pulmonaire du rat diabétique

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Membrane protein extraction and purification using styrene-maleic acid (SMA) co-polymer:effect of variations in polymer structure

The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst 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 to 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 weight (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.

Purification et identification d'une protéine de la membrane plasmique endothéliale : molécules d'adhésion de la microvascularisation pulmonaire du rat diabétique

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.