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Maltose Binding Protein (maltose + binding_protein)
Selected AbstractsExtra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coliFEBS JOURNAL, Issue 21 2002Sushil Prasad Sati The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal ,solubilization tag'. This system may allow large-scale production of those proteins that have a tendency to misfold during expression. [source] Glycosyltransferase Microarray Displayed on the Glycolipid LB MembraneADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6-7 2003Noriko Nagahori Abstract ,(1,4),Galactosyltransferase expressed as a fusion protein with maltose binding protein (MBP-GalT) was displayed specifically on a Langmuir,Blodgett (LB) membrane prepared by photopolymerization of maltotriose-carrying glycolipid (1) with 1,2-bis(10,12-tricosadiynoyl)- sn -glycero-3-phosphocholine (2). The catalytic activity of MBP-GalT on the LB film was directly monitored by the surface plasmon resonance (SPR) method using a GlcNAc-carrying water-soluble polymer (3) as an acceptor substrate. Highly sensitive sigmoidal-type signals were obtained upon the addition of the acceptor substrate in the presence of the donor substrate, UDP-galactose (UDP-Gal), while the binding of 3 was not detected in the absence of UDP-Gal. The intensities of the signals were dependent on the amount of immobilized MBP-GalT on the LB film, which was estimated from the images obtained by atomic force microscope (AFM). [source] A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reductionPROTEIN SCIENCE, Issue 5 2010Andrea F. Moon Abstract Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail. [source] WVD2 is a novel microtubule-associated protein in Arabidopsis thalianaTHE PLANT JOURNAL, Issue 6 2007Robyn M. Perrin Summary Arabidopsis WAVE-DAMPENED 2 (WVD2) was identified by forward genetics as an activation-tagged allele that causes plant and organ stockiness and inversion of helical root growth handedness on agar surfaces. Plants with high constitutive expression of WVD2 or other members of the WVD2-LIKE (WDL) gene family have stems and roots that are short and thick, have reduced anisotropic cell elongation, are suppressed in a root-waving phenotype, and have inverted handedness of twisting in hypocotyls and roots compared with wild-type. The wvd2-1 mutant shows aberrantly organized cortical microtubules in peripheral root cap cells as well as reduced branching of trichomes, unicellular leaf structures whose development is regulated by microtubule stability. Orthologs of the WVD2/WDL family are found widely throughout the plant kingdom, but are not similar to non-plant proteins with the exception of a C-terminal domain distantly related to the vertebrate microtubule-associated protein TPX2. in vivo, WVD2 and its closest paralog WDL1 are localized to interphase cortical microtubules in leaves, hypocotyls and roots. Recombinant glutathione- S -transferase:WVD2 or maltose binding protein:WVD2 protein bind to and bundle microtubules in vitro. We speculate that a C-terminal domain of TPX2 has been utilised by the WVD2 family for functions critical to the organization of plant microtubules. [source] Microbial bio-production of a recombinant stimuli-responsive biosurfactantBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009W. Kaar Abstract Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states. Biotechnol. Bioeng. 2009;102: 176,187. © 2008 Wiley Periodicals, Inc. [source] Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorptionBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005N. Abdullah Abstract Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His6 by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His6 to MBP was 16 h, as judged by SDS,PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions. © 2005 Wiley Periodicals, Inc. [source] Process intensification for the removal of poly-histidine fusion tags from recombinant proteins by an exopeptidaseBIOTECHNOLOGY PROGRESS, Issue 1 2010Wen-Hui K. Kuo Abstract This study describes the use of a hexa-histidine tagged exopeptidase for the cleavage of hexa-histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On-column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on-column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post-load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post-load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa-histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa-histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on-column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on-column exopeptidase cleavage makes the integration of the poly-histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly-histidine tag removal can now be combined with the on-column tag cleavage operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Adsorption of Cadmium Ion and Gallium Ion to Immobilized Metallothionein Fusion ProteinBIOTECHNOLOGY PROGRESS, Issue 6 2002Masaaki Terashima A fusion protein made from maltose binding protein (pmal) and human metallothionein (MT) was expressed using E. coli. The purified recombinant protein (pmal-MT) was immobilized on Chitopearl resin, and characteristics of pmal-MT for metal binding were evaluated. As expected from the tertiary structure of metallothionein, the pmal-MT ligand adsorbed 12.1 cadmium molecules per one molecule of the ligand at pH 5.2. The pmal-MT ligand also bound 26.6 gallium molecules per one molecule of the ligand at pH 6.5. Neither cadmium ion nor gallium ion bound to a control protein bovine serum albumin (BSA). Adsorption isotherms for both ions were correlated by Langmuir-type equations. Two types of binding sites have been elucidated on the basis of HSAB (hard and soft acid and base) theory. It was suggested that gallium ion specifically binds to amino acid residues containing oxygen and nitrogen atoms, while cadmium ion binds to specific binding sites formed by multiple cysteine residues. The pmal-MT ligand bound these metals in the concentration range of 0.2,1.0 mM, and the bound metal ions could be eluted under relatively mild conditions (pH 2.0). The pmal-MT Chitopearl resin was stable and could be used repeatedly without loss of binding activity. Thus, this new ligand would be useful for recovery of toxic heavy metals and/or valuable metal ions from various aqueous solutions. [source] Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinaseCLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2002G. O'Riordain Background In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue. Fruit-allergic patients represent one risk group for developing latex allergy. Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes. Objective To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02). Methods A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR. The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E. coli as a fusion protein to maltose binding protein. The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA. Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay. Results The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02). The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots. Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase. The growth of F. oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture. Conclusions Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein. [source] Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralisFEBS JOURNAL, Issue 14 2001Gerhard Greller We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 °C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein. [source] | |||||||||||||