Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000-6% (w/w) dextran T500 containing 10mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.     

Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.     

Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.     

A controlled pore glass bead assay for the measurement of cytoplasmic and nuclear glucocorticoid receptors

An assay for the quantitation of cytoplasmic and nuclear glucocorticoid receptors in lymphoid tissue has been developed using controlled pore glass (CPG) beads. Soluble receptor--3H-steroid complex (cytosol or nuclear extract) is adsorbed quantitatively within the crevasses of porous glass beads. Excess labeled steroid as well as most non-specifically bound steroid is easily washed away, leaving the hormone-receptor complex retained by the beads. Bound 3H-steroid is eluted with ethanol and measured for radioactivity. This procedure which is simple, rapid, and highly reproducible is carried out using frozen samples (stable for many months) containing as few as 1 X 10(7) cells. A comparison of the CPG assay to dextran coated charcoal and a whole cell assay demonstrates that CPG and dextran coated charcoal give equivalent measurements of cytosolic receptor concentration, while the CPG and whole cell assays provide equivalent values for total receptor content.     

Specific and non-specific affinities of the extracellular glucosyltransferase complex of Streptococcus mutans 6715

Glucosyltransferases (GTF) from different strains of streptococci exhibited different elution profiles when fractionated on insoluble-dextran affinity columns. The proportions of unadsorbed and adsorbed GTF were not related to their extent of stimulation by exogenous dextran, and GTF preparations exposed to, and freed from, clinical dextran prior to fractionation lost their ability to bind to the dextran columns. Different proportions of bound GTF were released by irrigation of columns with different concentrations of salt and clinical dextran, and the “specific” binding and release of GTF exhibited by a column possessing covalently linked, clinical dextran ligands was duplicated on a control column that did not possess the dextran ligands. These results, and the high affinity of GTF for hydrophobic alkyl (Shaltiel) ligands, demonstrate that ionic and hydrophobic properties of impure GTF aggregates may lead to erroneous characterization of the dextran affinity of some protein fractions. Fractionations on DEAE-Sepharose and on hydroxylapatite showed that the two dextran-dependant GTF activities (GTF-S and GTF-I) were present in the major enzyme fraction (Streptococcus mutans 6715) recovered from a Sephacryl S-200 affinity column. A minor, dextran-independent GTF was not adsorbed onto the Sephacryl column. The presence of SDS (0.005%) and Triton X100 (0.01%) stabilized GTF activity during gel filtration and improved the separation of GTF-S and GTF-I in hydroxylapatite fractionation of the highly aggregated enzyme. A comparable separation of the two enzyme forms on DEAE-Sepharose was achieved only if T10 dextran (10 mg/mL) was included with the detergent mixture in the column irrigant.     

Affinity chromatography of human anti-dextran antibodies isolation of two distinct populations

Affinity chromatography is a very efficient method for antibody purification. Two affinity chromatography supports were prepared to analyze the specificity of anti-dextran antibodies. Silica beads were grafted with native dextran or with functionalized dextran. The anti-dextran antibodies present in some human sera were analyzed by enzyme-linked immunosorbent assay method. These antibodies play an important role in severe dextran-induced anaphylactic reactions in humans by forming immune complexes with clinical dextran. The results indicated that two distinct populations of anti-dextran antibodies were purified from human serum, using dextran-coated silica beads. Elution from this support with an oligo-dextran of 4000 g/mol allowed the isolation of one population that only recognized native dextran as antigen. Functionalized dextran coated on dextran silica beads led to the purification, with a glycine-HCl buffer, of another subclass of antibodies that recognized substituted dextran derivatives. Furthermore, these antibodies could be useful tools for in vitro and in vivo investigations using dextran derivatives as bio-active polysaccharides.     

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.     

Measurement of genome wide DNA methylation by reversed-phase high-performance liquid chromatography

Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA.     

The Bead blot: a method for selecting small molecule ligands for protein capture and purification

We present a new method for selecting peptide ligands that are useful for protein purification, protein targeting and exploring protein-ligand interactions, and which requires no prior protein purification or derivatization. In the Bead blot, a complex mixture containing the target protein, for example, plasma, is incubated with a combinatorial library of peptide ligands synthesized on beads. The proteins are fractionated and purified on their respective ligands and the beads with their bound proteins are immobilized in a gel. The proteins are eluted from the ligands by capillary action and captured on a membrane so that their position on the membrane corresponds to the position of the beads in the gel. The protein is detected on the membrane, generating spots on an autoradiography film, the spots on the film are aligned with the beads in the gel, and the beads that bound the protein are recovered. The ligand on the bead(s) can be sequenced and synthesized at large scale for protein purification. The Bead blot can be completed in several hours with overnight pause steps if desired. On average, 5 prospective ligands are selected per 50,000 beads screened using this method. Unlike other ligand identification methods, the target protein does not have to be purified or labeled, and the Bead blot allows ligands for multiple proteins to be selected in a single experiment.     

Affinity of mouse immunoglobulin G subclasses for sialic acid derivatives immobilized on dextran-coated supports

High-performance liquid affinity chromatography is a powerful method for the purification of biological compounds owing to its specificity, rapidity and high resolution. In our laboratory, we develop chromatographic supports based on porous silica beads. However, in order to minimize non-specific interactions between the inorganic surface and proteins in aqueous solution, the silica beads are coated with modified dextran. As previously reported, many affinity ligands can be covalently grafted onto dextran-coated silica. In this study, N-acetylneuramic acid, which belongs to the sialic acid family and is present in immunoglobulin G (IgG) epitopes, is used as an active ligand. The interactions of this affinity support and IgG subclasses are analyzed. This immobilized ligand enables purification of IgG3 antibodies.     

Single-molecule imaging of interaction between dextran and glucosyltransferase from Streptococcus sobrinus

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.     

Poly(glycidylmethacrylate) brushes generated on poly(VBC) beads by SI-ATRP technique: Hydrazine and amino groups functionalized for invertase adsorption and purification

Crosslinked-poly(vinylbenzylchloride), poly(VBC), beads were prepared by suspension polymerization and poly(glycidylmethacrylate) was grafted by surface-initiated-atom radical polymerization (SI-ATRP) technique. Epoxy groups of the grafted poly(GMA) were reacted with hydrazine and ammonia to create an affinity binding sites. The hydrazine and amine functionalized poly(VBC-g-GMA) beads were used as an affinity support for adsorption of invertase from solution and yeast crude extract. The influence of pH, equilibrium time, ionic strength and initial invertase concentration on the adsorption capacities of both hydrazine and amine functionalized beads has been investigated. Maximum invertase adsorptions onto hydrazine and amine functionalized beads, were 86.7 and 30.4 mg/g at pH 4.0 and 5.5, respectively. The experimental equilibrium data fitted well to the Temkin isotherm model. Finally, the hydrazine functionalized poly(VBC-g-GMA) beads were used for the purification of invertase from crude yeast extract in a batch system and the purity of the eluted invertase from the hydrazine functionalized beads was determined as 92% by HPLC from single step purification protocol.     

New purification method for glucocorticoid receptors

In this report, we describe a new purification method for activated recombinant glucocorticoid receptor (GR) utilizing a cation-exchanger (Mono S) at pH8.4. This method is based upon a new finding that activated GR binds to both Mono Q and Mono S columns at the same pH. This method enables us to purify recombinant GR within 3 h. The purified GR represents more than 97% of the eluted proteins. Purified recombinant GR is able to bind specifically to a DNA fragment containing the glucocorticoid response element. Recombinant GR has no tag sequence that can be utilized for purification. Thus, this separation method is also applicable to purification of native GR.     

Quantitative evaluation of His‐tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

Histidine (His)‐tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of this study was to evaluate His‐tag procedure quantitatively and to compare it with immunoprecipitation using radiolabeled tristetraprolin (TTP), a zinc finger protein with anti‐inflammatory property. Human embryonic kidney 293 cells were transfected with wild‐type and nine mutant plasmids with single or multiple phosphorylation site mutation(s) in His‐TTP. These proteins were expressed and mainly localized in the cytosol of transfected cells by immunocytochemistry and confocal microscopy. His‐TTP proteins were purified by Ni‐NTA beads with imidazole elution or precipitated by TTP antibodies from transfected cells after being labeled with [³²P]‐orthophosphate. The results showed that (1) His‐tag purification was more effective than immunoprecipitation for TTP purification; (2) mutations in TTP increased the yield of His‐TTP by both purification procedures; and (3) mutations in TTP increased the binding affinity of mutant proteins for Ni‐NTA beads. These findings suggest that bioengineering phosphorylation sites in proteins can increase the production of recombinant proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Purification of human and avian influenza viruses using cellulose sulfate ester (Cellufine Sulfate) in the process of vaccine production

Affinity chromatography using sulfated, spherical cellulose beads (Cellufine Sulfate) was assessed for purification of influenza A and influenza B viruses. Recovery rates of viruses eluted from the beads were high for all tested virus strains. This method was also useful for removing chicken egg-derived impurities from allantoic fluids containing influenza viruses; the hemagglutination activity per amount of protein in the eluted sample was significantly higher than that in the applied sample. These results suggest that use of Cellufine Sulfate is a practical method for primary purification of influenza viruses in the process of influenza vaccine production.     

Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: generic, parallel, and scalable protein purification

We present the directed immobilization of recombinant antibody fragments as ligands for general immunoaffinity chromatography methods. It is based on fusion proteins of scFv fragments with several chitin-binding domains which can be immobilized directly from a crude bacterial lysate on inexpensive chitin beads for the purification of proteins without any gradient or detector. It has been used with a positive pressure manifold, allowing the parallel processing of 24 different samples on a milligram scale, as convenient as plasmid isolation. The method is demonstrated with several anti-protein antibodies. In addition, methods are presented of using an anti-His tag antibody either alone or directly coupled to IMAC to obtain very pure protein. As those methods are scalable, they should prove very useful in the parallel purification of natural and recombinant proteins on small scales (for proteomics), medium scales (for crystallography and NMR), and very large scales (for therapeutic proteins).     

Evaluation of viral contamination in a baculovirus expression system

Insect expression systems based on baculovirus are widely used for generating recombinant proteins. Here, the infectivity of baculoviruses under the physiological stresses of ‘freeze–thaw’ and sonication and the baculoviral contamination of recombinant proteins after protein purification were evaluated. Our findings suggest that Nonidet P‐40 (NP‐40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses. We therefore suggest that baculovirus is completely inactivated by NP‐40 treatment and that recombinant proteins are unlikely to be contaminated with infectious baculoviruses after their affinity purification.

     

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9 ± 2.6 mg/g beads. Adsorbed trypsin was eluted by using (0.1 M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.     

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.