Subsequent Size Exclusion Chromatography Research Articles

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Purification and In Vitro Functional Analysis of Glutamate Receptor

Ionotropic glutamate receptors are predominantly localized to neuronal synapses and are principal mediators of excitatory neurotransmission in the brain. Receptor function is regulated by multiple factors including ligands, ions, pH, posttranslational modification, subunit composition, RNA editing, and elements of the lipid environment. Analysis of channel function in native membranes has provided much quantitative information on these various regulatory features, however, investigation of lipid regulation is seriously hampered by an inability to control membrane composition in living cells. Although an inhibitory effect of free polyunsaturated fatty acids on GluK2(R) activation has been demonstrated, the specific bulk lipid requirements for normal channel operation, as well as susceptibility to fatty acid modulation, are unknown. To address this, we aim to purify rat kainate receptor 2 (GluK2), to reconstitute the purified protein, and to assess channel function under different defined lipid environments. Using Saccharomyces cerevisiae as an expression host, we have been able to express wild type GluK2 with either Gln or Arg at the editing site, and with different tags. In addition, we have expressed modified forms of GluK2 including an amino terminal domain (ATD) deletion mutant, and a double point mutation (Y590C/L572C) that allows disulfide crosslinking between ligand binding domain (LBD) dimers. We have succeeded in purifying wild type GluK2(R) through Flag affinity and subsequent size exclusion chromatography after solubilizing the protein using the detergent Foscholin-14 (F14). A major band at the expected monomer size of ∼100kDa and several bands at lower molecular weights are resolved on 1D SDS PAGE and are confirmed to be GluK2 through mass spectrometry (MS) analysis. MS analysis also reveals that a fraction of the heterologously expressed protein in yeast maintains a signal sequence at the N-terminus and is phosphorylated on a presumed extracellular residue, indicating misoriented topology.

Isolation and characterization of immuno-stimulating polysaccharide in Korean black raspberry wine

To characterize polysaccharides existing as soluble form in Korean fruit wines, polysaccharides were isolated from Korean black raspberry wine, and their immuno-stimulating activities were examined. A complex pectic polysaccharide, RB-2 was purified from the crude polysaccharide (RB-0) in raspberry wine by subsequent size-exclusion chromatography. RB-2 had relatively potent macrophage activity and splenocyte mitogenic activity in vitro. The intravenous administration of RB-2 significantly augmented the cytotoxicity of natural killer cells against Yac-1 tumor cells. RB-2 consisted of 15 monosaccharides including rarely observed sugars in general polysaccharides, such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3-deoxy-D-manno-2-octulosonic acid, and 3-deoxy-d-lyxo-2-heptulosaric acid. Methylation analysis indicated that RB-2 comprised at least 20 different glycosyl linkages such as 3,4-linked fucose, 2,3,4-linked rhamnose, 3′-linked apiose, and 2,3,3′-linked apiose, characteristic of rhamnogalacturonan II (RGII). RB-2 was eluted as two peaks having a high (9,000 Da) and a low molecular mass (5,000 Da) on high performance size exclusion chromatography, and the peak of high molecular mass was down shifted to the low molecular mass under the acidic pH conditions less than 2.0. Results indicated higher molecular weight polysaccharide in RB-2 was mainly presented as an RG-II dimer, and Korean black raspberry wine contains selected polysaccharides, which provide immunostimulating activities beneficial to human health.

Recombinant production of bioactive human TNF-α by SUMO-fusion system – High yields from shake-flask culture

Tumor necrosis factor (TNF-α) inhibitors, used for the treatment of common inflammatory diseases, currently belong among the most important biotechnologically produced pharmaceuticals. So far four TNF-α antagonists have been approved by regulatory authorities for defined subsets of applications. Furthermore, numerous approaches are being taken to develop new protein-based pharmaceuticals and to broaden their application areas in the treatment of TNF-α -related diseases. Both the fundamental understanding of disease-related TNF-α activity and the subsequent development of corresponding drug candidates demand the availability of large amounts of TNF-α as a bioactive protein. We have therefore established a protocol for the rapid high-level synthesis of recombinant human TNF-α in Escherichia coli shake-flask cultures and the subsequent purification of the mature protein. Using the advantages of SUMO-fusion technology we were able to produce protein with an authentic N-terminus in high yield. Two immobilized metal ion-affinity chromatography steps with a protease cleavage step in between and subsequent size-exclusion chromatography were utilized to purify the protein. The protein was obtained from the last chromatography step as a trimer, while purity was at least 96% as estimated by SDS–PAGE. The identity of the protein was confirmed by MALDI-TOF mass spectrometry. Recombinant mature TNF-α was correctly folded as assessed by CD spectroscopy and its biological activity was confirmed by an L929 cell assay.

Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography

Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2

Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.

Downstream processing of MDCK cell-derived equine influenza virus

A microcarrier-based process was used to produce equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93) in Madin Darby Canine kidney (MDCK) cells. The virus was purified in a sequence of downstream processing steps comprising of depth filtration, inactivation, ultrafiltration (UF) and gel filtration. In the ultrafiltration step, the hemagglutinin (HA) was recovered to 100%. A high increase of neuraminidase (NA) activity indicated the removal of some inhibitory compounds during this step. At the same time, the level of contaminating proteins and DNA was reduced by more than 88%. In the subsequent size exclusion chromatography (Sepharose CL 2B), the recovery of HA and NA in the “virus peak” was 37.8 and 59.8%, respectively compared to the concentrated feed material. Inconsistencies in the overall mass balance for HA and NA (70.0 and 69.2%) during gel filtration indicated non-specific interactions of the inactivated virus to the gel matrix which is supported by a HA recovery of about 50% in shake flask experiments performed as a control. Overall 35.8% of HA and 291.6% of NA were recovered. More than 95.7% of the host cell proteins and 98.7% of the host cell DNA were removed during downstream processing.

Two forms of membrane-bound sphingosine kinase in Tetrahymena and activity changes during growth and the cell cycle.

Sphingosine kinase is responsible for the formation of sphingosine-1-phosphate, a sphingolipid mediator with important roles in numerous physiological processes. The sphingosine kinase activity of Tetrahymena pyriformis was recovered predominantly in the particulate fraction and it could be solubilised in 1% beta-octylglucoside. Anion-exchange chromatography resolved the beta-octylglucoside-solubilised sphingosine kinase activity into two peaks corresponding to proteins of Mr 140,000 and 80,000 respectively, as determined by subsequent size exclusion chromatography on Superdex 200. N,N-dimethylsphingosine did not inhibit the sphingosine kinase activity in either fraction, whereas D,L-threo-dihydrosphingosine inhibited sphingosine phosphorylation by the Mr 80,000 kinase but had no effect on the Mr 140,000 kinase activity. The activities also showed different stimulatory responses to Triton X-100 or NaCl. Overall, the results suggest the existence in Tetrahymena of two distinct membrane-associated sphingosine kinases. The kinase activity determined at the different culture stages showed a transient elevation at the mid-logarithmic phase. Further, the sphingosine kinase activity was examined during the synchronous cell division induced by cyclic heat treatments in T. pyriformis. We report for the first time that the sphingosine kinase activity greatly increased at 30 to 45 min after the end of heat treatment prior to the synchronous cell division (75 min), suggesting that the activity changes were associated with the cell cycle and that the up-regulated sphingosine kinase activity would be required for the initiation of the oncoming synchronous cell division in Tetrahymena cells.

Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium

This study deals with the effect of methyl esters, acetyl groups, and neutral sugar side-chains on the gelation properties of sugar beet pectin with Ca 2+. Sugar beet pectin was treated in the presence of Ca 2+ with the enzymes pectin methyl esterase (PE), pectin acetyl esterase (PAE), rhamnogalacturonan acetyl esterase (RGAE), arabinofuranosidase B (AF) and rhamnogalacturonase (RGase) in various combinations. Addition of RGAE plus PE or PAE plus PE to the pectin–Ca 2+ mixture significantly increased the release of acetyl groups and methyl esters, in comparison to the addition of only PE or PAE. This indicates that PE activity is hindered by the presence of acetyl groups both in the ‘smooth’ and in the `hairy’ regions. Also the PAE activity is hindered by the presence of methyl groups in the ‘smooth’ regions. Treatment with PAE plus PE led to a stiffer gel, as determined by the storage modulus ( G′), than treatment with PE alone, while RGAE plus PE did not improve the gel forming properties. Addition of only PAE to the pectin–Ca 2+ mixture did not result in gel formation. A lower stiffness of the gel was found when RGase combined with RGAE and PE were added to the pectin–Ca 2+ mixture, in comparison to treatment with PE alone. Addition of AF plus PE to the pectin–Ca 2+ mixture gave similar rheological effects as treatment with only PE. A fraction representing the ‘smooth’ homogalacturonan regions, which was obtained after treatment of the beet pectin with RGase and subsequent size-exclusion chromatography, was also able to form a gel with Ca 2+ and PE. However, the gel formation was much slower, and the stiffness of the gel was lower than when the parental extract was used. Also with the modified pectin the treatment with PAE plus PE gave an increased stiffness of the gel in comparison to PE alone.

Selenium speciation in human milk with special respect to quality control.

Selenium- (SE) organo compounds of pooled human milk (7th-14th d after delivery) were separated by centrifugation and subsequent size-exclusion chromatography (SEC) as described in ref. (1). The SEC fractions were used for Se determinations by electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS) in parallel to identification procedures of the organic ligands by two different capillary zone electrophoresis (CZE) methods. Further, the combination of isotachophoresis- (ITP) CZE with ETV-ICP-MS was used for final identifications. Mass balances were carried out at each analytical step for quality assurance. Reinjection experiments were performed to check the stability of Se-organo compounds during the analytical procedure. These quality-control experiments showed that no species transformations took place during the analytical procedure, and the Se species were native in human milk. The identification and quantification of organic ligands were clear and resulted in values of 2 (+/- 0.2) mg/L GSH/GSeH, 2 (+/- 0.22) mg/L cystamine/Se-cystamine, 4 (+/- 0.4) mg/L cystine/ Se-cystine, and 1 (+/- 0.18) mg/L methionine/Se-methionine. Unfortunately, a differentiation between sulfur (S) and Se analogs was not possible with the applied CE methods. The Se values per organic ligand were determined as 2.5 (+/- 0.23) mg/L associated with GSH (as GSeH), 3.1 (+/- 0.31) mg/L associated with cystamine (as Se-cystamine), 5.2 (+/- 0.4) mg/L associated with cystine (as Se-cystine), and 1 (+/- 0.1) mg/L associated with methionine (as Se-methionine).

The insulin-like growth factor system in human peritoneal fluid: its effects on endometrial stromal cells and its potential relevance to endometriosis.

Peritoneal fluid (PF) lines the abdomen and pelvis and is believed to contain growth factors that stimulate endometriosis, a benign gynecological condition associated with pelvic pain and infertility, in which endometrial cells proliferate and differentiate on the pelvic peritoneum, outside of their normal location within the uterus. In this study, we examined the insulin-like growth factor (IGF) system in seven paired samples of PF and serum from normally cycling women and examined the mitogenic potential of this fluid on cultured endometrial stromal cells. IGF-I, IGF-II, and IGF-binding protein-1 (IGFBP-1), -2, -3, and -4 were identified in PF by immunoassays. PF IGF levels, determined by RIA, were approximately 60% of paired serum levels, and PF levels of IGFBP-2 and IGFBP-3, determined by Western ligand blotting and RIA, respectively, were approximately half of their serum concentrations. IGFBP-4 was barely detectable by Western ligand blotting in PF, and levels of IGFBP-1, determined by immunoassay, were not appreciably different in PF and serum. Incubation of [125I]IGF-II with serum and PF and subsequent size-exclusion chromatography at neutral pH revealed approximately equal incorporation of radiolabel in the IGFBP regions of 150 and 44 kilodaltons (kDa) in serum and primarily in the 44-kDa region in PF. RIA of IGFBP-3 in the IGFBP regions of column effluent revealed that the majority of IGFBP-3 was in the 150-kDa region in both serum and PF, suggesting the presence of the ternary complex in PF. Western ligand blotting of column effluent samples revealed 37-/43-kDa IGFBP-3 primarily in the 150-kDa complex in serum and a marked reduction in the amount of the 37-/43-kDa IGFBP in PF. Western immunoblotting of column effluent with IGFBP-3 antiserum revealed immunoreactive IGFBP-3 forms of 37-43 kDa (major) and 28 kDa (minor) in serum and almost exclusively the 28-kDa band in PF, suggesting that IGFBP-3 in PF may be proteolytically processed. The presence of an IGFBP-3 protease was confirmed using [125I]IGFBP-3 as substrate and was not appreciably present in paired serum samples. Inhibitor profiles demonstrated that this protease is a metal-independent serine protease, and its approximate relative molecular mass was estimated to be 69 kDa, determined by size-exclusion chromatography. The mitogenic potential of IGF peptides and PF was assessed on cultured endometrial stromal cells to test the hypothesis that IGFs in PF may stimulate the growth of endometrium in the pelvic cavity, for example in the disorder of endometriosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Composition distribution separation of methyl methacrylate-methacrylic acid copolymers by normal-phase gradient elution high-performance liquid chromatography

The characterization of copolymers requires the determination of chemical composition distribution in addition to molecular mass ( M) distribution. Techniques commonly applied to copolymer analysis provide either average bulk composition or a convolution of M and composition distributions. The application of gradient elution adsorption chromatography, under appropriate conditions, allows the separation of copolymers based solely upon their chemical composition in a M-independent mode. Separation independent of M requires the elimination of size-exclusion effects with the porous adsorbent and the minimization of precipitation contributions. For separation over a wide copolymer composition range, the relative contributions of adsorption and precipitation retention mechanisms change with solvent composition. Under gradient elution conditions where adsorption retention dominates, composition fractionation can be combined with subsequent size-exclusion chromatography to provide a complete composition/ M map of the copolymer.

Formation of aggregates of tryptic fragments derived from the carboxyl-terminal half of pea phytochrome and localization of the site of contact between the fragments by amino-terminal amino acid sequence analysis.

Limited trypsinization of large pea (Pisum sativum cv. Alaska) phytochrome and subsequent size-exclusion chromatography (SEC) in 0.1 M Na phosphate, pH 7.8, yielded a high-molecular-mass aggregate of tryptic fragments of phytochrome. Further SEC in 0.1 M Tris-HCl, pH 7.5, plus various concentrations of NaSCN, indicated that the tryptic-fragment complex contained an aggregate of 7 fragments of molecular mass from 38 to 55 kDa. The amino-terminal sequence of each fragment was determined from the samples electroblotted from sodium dodecylsulfate polyacrylamide gels onto polyvinylidene difluoride membranes, in order to localize the various fragments on the phytochrome polypeptide chain. All of the 7 fragments in the aggregate were found to be derived from the carboxyl-terminal half of phytochrome. A portion of the polypeptide chain (from Ala-752 to Arg-1000) common to all the tryptic fragments has been assigned as the site(s) of contact of the fragments. The tryptic-fragment complex, as well as large phytochrome itself, has been shown by SEC to dissociate to monomers in 2 M NaSCN. The result indicates that the main force involved in maintaining the complex and in contacts between monomers of phytochrome is non-ionic in nature. Relationship between the contact site(s) of the tryptic-fragment complex and large phytochrome monomer is discussed.

The somatomedin-binding protein isolated from a human hepatoma cell line is identical to the human amniotic fluid somatomedin-binding protein

Serum-free medium conditioned by the human hepatoma cell line HEP G2 was shown to contain a somatomedin-binding protein with a relative molecular mass of about 35,000. This binding protein was purified to homogeneity by the use of immunoaffinity chromatography and subsequent size exclusion chromatography. Antibodies for the immunoaffinity step were raised in rabbits against a previously isolated human amniotic fluid somatomedin-binding protein. The total composition and N-terminal amino acid sequence showed the protein to be identical to the binding protein from human amniotic fluid. Both have the N-terminal structure Ala-Pro-Trp-Gln-. The HEP G2 cell line offers a useful model to study the regulation of the synthesis and secretion of human somatomedin-binding proteins.