Immobilized Metal Ion Chromatography Research Articles

Study on the transient expression of infectious bronchitis virus spike protein in Nicotiana benthamiana leaves

Infectious bronchitis virus (IBV) is considered as one of the main causes of economic loss in chicken farms worldwide, especially in poultry producing nations. This virus, a member of the Coronaviridae family, is classified into different genotypes based on its surface spike glycoproteins which also play important roles in cell attachment and immune response. Due to continual genetic mutation of IBV, attenuated and inactivated vaccines show decreased effectiveness or lack of cross – protection; thus ongoing studies focus on the development of new generation vaccines to prevent new IBV outbreaks. This paper describes our study on expression of the antigenic region in S1 subunit of the spike in Nicotiana benthamiana. Two expression vectors carrying either S1 or receptor binding domain (RBD) coding gene (pCB301-S1 and pCB301-RBD) were constructed and transformed into tobacco leaves by agroinfiltration method. The RBD showed a clearly higher level of expression compared to the whole S1. Purification of RBD by immobilized metal ion chromatography and size exclusion chromatography represented a mixture of monomer, dimer and trimer glycoprotein with expected sizes in Western blot. In summary, this study demonstrated our primary success in establishing an expression model that could be used to investigate plant-based IBV recombinant vaccine.

Hydrophilic magnetic host-guest Ti-phenolic networks: a promising material for the highly sensitive enrichment of glycopeptides and phosphopeptides.

Protein glycosylation and phosphorylation are the two most important post-translational modifications, which play a vital role in physiological and pathological processes. Before the comprehensive characterization of glycoproteome/phosphoproteome through the mass spectrometry (MS) technique, it is necessary to perform a highly specific enrichment procedure because glycoproteins/phosphoproteins inherently occur in low abundances. Herein, we have reported a novel magnetic β-cyclodextrin-based host-guest Ti-phenolic network material, focusing on the simultaneous enrichment of glycopeptides/phosphopeptides via hydrophilic interaction chromatography and immobilized metal ion chromatography. Ti ions and glutathione-derived adamantine were introduced by metal-phenolic interactions and host-guest interactions. The material possesses biocompatibility, good hydrophilicity, strong magnetic response, metal chelation effect, and demonstrates an excellent enrichment ability towards glycopeptides/phosphopeptides. Combined with MS detection, high sensitivity (0.035/0.01 fmol for IgG/β-casein) and good reusability (6 times) were achieved. In addition, its outstanding specificity was validated in quantities as low as 500 : 1 : 1 for BSA : IgG : β-casein (m/m/m). Benefiting from these merits, the adsorbent material was successfully used for the simultaneous enrichment of phosphopeptides/glycopeptides from human serum and HeLa cell lysate and can be expected to exhibit great applicability for precious and small amounts of biosamples in the study of glycoproteomics/phosphoproteomics.

Producción y caracterización de una esterasa extracelular termoestable de Aspergillus niger

A strain of Aspergillus niger NRRL 337 degrade agro-industrial waste i.e wheat bran in solid state fermentation and produce esterase enzyme. Maximum enzyme activity 15.2 U/ml was produced after 72 h of incubation at 30˚C. The protein was partially purified by performing ammonium sulfate precipitation and immobilized metal ion chromatography (IMAC) technique. SDS-PAGE analysis showed molecular weight of esterase was 70 kDa. Effects of different nitrogrn and carbon sources with different concentrations were also studied. Partially purified enzyme was active at pH of 6.0 for up to 3 h and at temperature of 70˚C for 1 h. Ca2+, Mg2+, Na+1, and K+1 play an important role in increasing enzyme activity. The residual activity of esterase was decreased in the presence of organic solvents. SDS, β-mercaptoethanol, and DMSO are good inhibitors for esterase enzyme. Under these conditions, partially purified esterase with specific activity of 114.20 U/mg and 47.58 purification fold was obtained. The data achieved from this work are important in terms of heat stability and pH of esterase and giving a new enzyme source for industrial applications.

A versatile tool in controlling aggregation and Ag nanoparticles assisted in vitro folding of thermally denatured zDHFR

BackgroundProteins have tendency to form inactive aggregates at higher temperatures due to thermal instability. Maintenance of thermal stability is essential to gain the protein in sufficient quantity and biologically active form during their commercial production. MethodsBL21-DE3 Rosetta E. coli cells which contains plasmid pET43.1a vector was used for producing zDHFR protein commercially. The purification of N-terminal Histidine tagged zDHFR was performed by Immobilized Metal Ion chromatography (IMAC). Investigations were performed in existence and non existence of Silver nanoparticles (AgNPs). The inactivation kinetics of zDHFR in existence and non existence of AgNPs were monitored over a range of 40–80 °C as monitored by UV–Visible absorption spectroscopy. ResultsThe protein completely lost its activity at 55 °C. Kinetics of inactivated zDHFR follows first order model in presence and absence of AgNPs. Decrease in rate constant (k) values at respective temperatures depicts that AgNPs contribute in the thermostability of the protein. AgNPs also assists in regaining the activity of zDHFR protein. ConclusionsAgNPs helps in maintaining thermostability and reducing the aggregation propensity of zDHFR protein. General significanceResult explains that AgNPs are recommended as a valuable system in enhancing the industrial production of biologically active zDHFR protein which is an important component in folate cycle and essential for survival of cells and prevents the protein from being aggregated.

Characterization and Expression Profiling of Recombinant Parathyroid Hormone (rhPTH) Analog 1–34 in Escherichia coli, Precise with Enhanced Biological Activity

Human truncated parathyroid hormone [hPTH (1–34)], a peptide hormone which accelerated the research interest towards the theraputical applications. This study outlines the effective expression of [hPTH (1–34)] in Escherichia coli and the recuperation of highly soluble truncated PTH from the fusion protein by proteolytic digestion. Successful expression of glutathione S-transferase (GST) fusion protein was achieved by incorporating truncated PTH with an enzymatic cleavage site into the “N” terminal GST of pGEX-4T-3 expression vector. Under the optimized condition, we achieved more than 120 mg of pure hPTH (1–34) per liter of bacterial culture, with an overall yield of 39%. Purification process was carried out through immobilized metal ion chromatography and membrane filter to produce maximum purity. Physical characterization using western blot analysis showed that the extracted truncated PTH is intact and reacts with anti-PTH antibodies. In vitro analysis of PTH stimulated adenylyl cyclase activation in UMR 106 cells confirmed biological activity for purified protein. We believe this vector and production methodology represents a generally applicable tool for the generation of recombinant peptides.

Detection of histidine-rich glycoprotein and fibrinogen with nickel-enzyme conjugates: Purification of rabbit HRG

Nickel-bound alkaline phosphatase and peroxidase enzymes were used to investigate nickel binding to plasma proteins. Rabbit plasma dilutions to 25,000 were positive by ELISA, while Western blot analysis showed a prominent reaction with histidine-rich glycoprotein (HRG)1 and lower reaction with fibrinogen (Fgn). To confirm their identities, purified HRG and Fgn were demonstrated to react with the nickel-bound enzymes by Western analysis. With disulfide bonds reduced, HRG and Fgn α-chain reactions were demonstrated. HRG reactions were shown in other species, including human, bovine, chicken and guinea pig, demonstrating general applicability of the detection method. To enhance the purification of rabbit HRG, ammonium sulfate fractionation, immobilized metal ion chromatography and ion-exchange chromatography were optimized. Purified HRG contained trace components larger than HRG that reacted with nickel-enzymes and also with an antibody to HRG by Western analysis, confirming the trace components are related to HRG. These results demonstrate the utility of nickel-enzymes together with antibodies to detect HRG.

Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis.

Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors.

Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for gluco- and galacto- substituted mannans and its calcium induced stability.

This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (K a = 14.3×104 M−1), carob galactomannan (K a = 12.4×104 M−1) and negligible association (K a = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (K a = 41×104 M−1) and carob galactomannan (K a = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.

Recombinant Production, Isotope Labeling and Purification of ENOD40B: A Plant Peptide Hormone

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.

Hierarchical Fe-ZSM-5 zeolite monolithic column for enhanced phosphorylated protein immobilization and identification

A hierarchical Fe-ZSM-5 zeolite monolithic column was fabricated for immobilization of low abundance phosphoprotein through specific coordination between heteroatomic Fe in the framework of the zeolite with the phosphate group in the micro-environment of the protein. Compared to traditionally immobilized metal ion chromatography, this method could be directly manipulated and handily adapted for phosphoproteomics identification.

A proteomic approach to identification of plutonium-binding proteins in mammalian cells

Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry was employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu(4+)-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore™ analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase

The use of Escherichia coli protein expression systems has many benefits, including the ease of propagation, amounts of protein that can be generated and cost. However, this host has some drawbacks due to difficulties in the production of soluble foreign proteins with their alternate codon usage bias, reductive cytoplasmic environment and lack of complex post-translational modifications. We have designed a novel fusion protein tag derived from the sequence of sortase (SrtA) which we have named Solubility 'eNhancing'Ubiquitous Tag (SNUT). Here we demonstrate its application and effectiveness as an N-terminal fusion tag for the expression and purification of proteins that could not be effectively produced with other tags. We show this tag can be utilized for the purification of proteins through both native and refolding immobilized metal ion chromatography and in combination with an anti-SNUT monoclonal antibody, can also be used as a detection tag. This tag may prove useful in circumventing expression and purification issues with the production of proteins in bacterial expression hosts.

Thiol protection in membrane protein purifications: A study with phage holins

The lambda holin, or S105, is a small cytoplasmic membrane protein that controls the timing of host lysis. Using thiol-specific reagents, we determined that the single cysteine residue within S105 was heterogeneously modified during membrane extraction and subsequent immobilized metal ion chromatography. Here we describe the use of a specific and reversible thiol reagent, 2,2′-dithiodipyridine, to generate purified protein with its cysteine residues in the native thiol state. The 2,2′-dithiodipyridine protection protocol was also successfully used for another unrelated holin, S 2168, and should be generally useful for the purification of membrane proteins.

General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 --> Gly and Arg-50 --> Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 --> Val and Gly/Ala-52 --> Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity.

Expression, purification and characterization of BGERII: a novel pan-TGF inhibitor

Transforming growth factor beta (TGFbeta) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFbeta isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFbeta type II (RII) and betaglycan (BG) have been utilized as TGFbeta inhibitors, their antagonistic potency against different TGFbeta isoforms varies considerably because RII does not appreciably bind to TGFbeta2 whereas BG binds weakly to TGFbeta1 and TGFbeta3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BG(E)) and the extracellular domain of RII. The fusion protein (named BG(E)RII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFbeta1 and TGFbeta3 than a commercially available soluble RII and to TGFbeta2 than a commercially available soluble BG. More significantly, whereas BG(E) or RII alone showed no antagonistic activity towards TGFbeta2, BG(E)RII inhibited the signaling of both TGFbeta1 and TGFbeta2 in cell-based assays including TGFbeta-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFbeta-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BG(E)RII was found to have greater activity than other potent TGFbeta inhibitors in blocking the signaling of TGFbeta1 and TGFbeta3. Thus, BG(E)RII is a potent pan-TGFbeta inhibitor in vitro and has potential for blocking TGFbeta-induced pathogenesis in vivo.

Effect of Skin Contact and Pressure on the Composition of Sauvignon Blanc Must

Early white winemaking operations are known to affect the extraction of grape skin compounds into the juice fraction, which will dictate their concentration in the resulting wine. Grape skin contact and the amount of pressure applied during grape pressing affect the extraction of varietal aromas located in the skins. Compounds such as the polyphenols and glutathione, with antioxidant properties involved in juice oxidation processes and white wine stability, are also affected. The present study evaluates how grape skin contact and the amount of pressure applied during grape pressing affect the levels of S-(3-hexan-l-ol)cysteine (3MH-S-cys, a key grape-derived precursor to the volatile thiol 3-mercapto-hexanol (3MH), which is reminiscent of passion fruit aroma); 2-methoxy-3-isobutylpyrazine (IBMP, with a capsicum-like descriptor); phenolic compounds; and glutathione in Sauvignon Blanc juice. The study was conducted using grapes obtained from commercial Marlborough (New Zealand) vineyards, using both commercial and laboratory grape-processing procedures. Immobilized metal ion chromatography was used to isolate the 3MH- S-cys precursor from the juices. The isolated precursor was then volatilized by trimethylsilylation and analyzed using gas chromatography/mass spectrometry (GC/MS). IBMP was analyzed by GC/MS after solvent extraction, and a high-performance liquid chromatography method was used for the quantification of phenolic compounds and glutathione. 3MH- S-cys levels were seen to increase in juice fractions obtained from a winery press operating at higher pressures. The increase was attributed to the cumulative effect of longer skin contact time and the amount of pressure applied. The highly water-soluble IBMP was less affected by the amount of pressure applied during commercial grape pressing. Additional information was generated by the specific assessment of skin contact and applied pressure during grape pressing in a laboratory trial. In this trial, a long (32 h) skin contact time resulted in a greater release of varietal aroma compounds, 3MH- S-cys, and IBMP into the juice, and the concentration was further raised by increasing the pressure applied during pressing. However, for both experiments, the extraction of the varietal aroma compounds was offset by a clear increase in the juice oxidative potential, seen by a decline in glutathione content, a natural grape antioxidant, and an increase in particular oxidizeable polyphenol compounds, which may cause the must or wine to brown and lead to a loss of varietal aromas.

Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus

The question whether proteins fused to β1,4galactosyltransferase (β4GalT-1) influence the biocatalytic properties of the glycosyltransferase has not been addressed so far. In the present study we have chosen a novel approach to express the gene encoding for human β4GalT-1 from placenta in an N-terminal fusion with the pre-propeptide of the lipase from Staphylococcus hyicus. The pre-propeptide provides a secretion signal in Escherichia coli and was reported to protect fused proteins against proteolytic degradation. Expression of the fusion protein was challenged with an almost full-length version of human β4GalT-1 including parts of the signal anchor and the stem region (propeptide-natβ4GalT-1) and the full catalytic domain (His 6propeptide-catβ4GalT-1), respectively. We demonstrate that the fusion protein in propeptide-natβ4GalT-1 is cleaved off during purification using immobilized metal ion chromatography IMAC, most probably catalyzed by the immobilized Zn 2+ ions. Cleavage can be avoided by deletion of five C-terminal amino acids of the propeptide and the stem region yielding His 6propeptide-catβ4GalT-1. Kinetic data reveal that both enzyme constructs possess specific biocatalytic characteristics when compared to a recombinant luminal β4GalT-1 construct. The catalytic efficiency towards more hydrophobic acceptor glycosides, e.g. benzyl 2-acetamido-2-deoxy-β- d-glucopyranoside and p-nitro phenyl 2-acetamido-2-deoxy-β- d-glucopyranoside, and the N-glycans of IgG from rat is significantly increased. In summary, the His 6propeptide-catβ4GalT-1 is very useful for biocatalytic applications involving hydrophobic acceptor glycosides and the propeptide-natβ4GalT-1 is able to glycosylate glycoproteins in an efficient way.

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

The enzyme dextransucrase (sucrose:1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2.4.1.5) catalyses the synthesis of exopolysaccharide, dextran from sucrose. This class of polysaccharide has been extensively exploited in pharmaceutical industry as blood volume expander, as stabiliser in food industry and as a chromatographic medium in fine chemical industry because of their nonionic nature and stability. Majority of the dextrans are synthesized from sucrose by dextransucrase secreted mainly by bacteria belonging to genera Leuconostoc, Streptococcus and Lactobacillus. Bulk of the information on purification of extracellular dextransucrase has been generated from Leuconostoc species. Various methods such as precipitation by ammonium sulphate, ethanol or polyethylene glycol, phase partitioning, ultrafiltration and chromatography have been used to purify the enzyme. Purification of dextransucrase is rendered difficult by the presence of viscous dextran in the medium. However, processes like ultra-filtration, salt and PEG precipitation, chromatography and phase partitioning have been standardized and successfully used for higher scale purification of the enzyme. A recombinant dextransucrase from Leuconostoc mesenteroides B-512F with a histidine tag has been expressed in E. coli cells and purifi ed by immobilized metal ion chromatography. This review reports the available information on purifi cation methods of dextransucrase from Leuconostoc mesenteroides strains.