Surface Properties of Protein-Polyelectrolyte Solutions. Impact of Polyelectrolyte Hydrophobicity.

The strong influence of an amphiphilic polyelectrolyte, poly(,-diallyl--hexyl--methylammonium chloride), on the surface properties of solutions of globular proteins (lysozyme, β-lactoglobulin, bovine serum albumin, and green fluorescent protein) depends on the protein structure and allows elucidation of the contribution of hydrophobic interactions in the protein-polyelectrolyte complex formation at the liquid-gas interface. At the beginning of adsorption, the surface properties are determined by the unbound amphiphilic component, but the influence of the protein-polyelectrolyte complexes of high surface activity increases at the approach to equilibrium. The kinetic dependencies of the dilational dynamic surface elasticity with one or two local maxima give a possibility to distinguish clearly between different steps of the adsorption process and to trace the formation of the distal region of the adsorption layer.

Recombinant Multi-l-Arginyl-Poly-l-Aspartate (Cyanophycin) as an Emerging Biomaterial.

Multi-l-arginyl-poly-l-aspartate (MAPA) is a non-ribosomal polypeptide which synthesis is directed by cyanophycin synthetase, and its production can be achieved using recombinant microorganisms carrying the cphA gene. Along its poly-aspartate backbone, arginine or lysine links to each aspartate via an isopeptide bond. MAPA is a zwitterionic polyelectrolyte full of charged carboxylic, amine, and guanidino groups.

Altering the substrate specificity of recombinant l-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 to favor d-allose production.

l-Rhamnose isomerase (l-RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l-RhI (TsRhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R TsRhIs can increase the preference against d-allose in comparison with the wild-type enzyme.

Wound Healing Attributes of Polyelectrolyte Multilayers Prepared with Multi-l-arginyl-poly-l-aspartate Pairing with Hyaluronic Acid and γ-Polyglutamic Acid.

Biodegradable multi-l-arginyl-poly-l-aspartate (MAPA), more commonly cyanophycin, prepared with recombinant Escherichia coli contains a polyaspartate backbone with lysine and arginine as side chains. Two assemblies of polyelectrolyte multilayers (PEMs) are fabricated at three different concentration ratios of insoluble MAPA (iMAPA) with hyaluronic acid (iMAPA/HA) and with γ-polyglutamic acid (iMAPA/γ-PGA), respectively, utilizing a layer-by-layer approach. Both films with iMAPA and its counterpart, HA or γ-PGA, as the terminal layer are prepared to assess the effect on film roughness, cell growth, and cell migration.

Reversible Self-Assembly Nanovesicle of UCST Response Prepared with Multi-l-arginyl-poly-l-aspartate Conjugated with Polyethylene Glycol.

Multi-L-arginyl-poly-L-aspartate (MAPA), also known as cyanophycin, containing a backbone of polyaspartate with arginine and lysine as side chains, was prepared with recombinant Escherichia coli. The insoluble part (iMAPA) was conjugated with polyethylene glycol (PEG) at two different levels, high (iMAPA(PEG)h) and low (iMAPA(PEG)l). Both levels of conjugation exhibited UCST (upper critical solution temperature)-type responses in the pH range of 3-10 at a concentration of 2 mg/mL.

Characterization of a recombinant d-allulose 3-epimerase from Agrobacterium sp. ATCC 31749 and identification of an important interfacial residue.

d-Allulose 3-epimerase (DAEase) catalyzes the epimerization between d-fructose and d-allulose. We had PCR-cloned and overexpressed the gene encoding Agrobacterium sp. ATCC 31749 DAEase (AsDAEase) in Escherichia coli.

Overexpression and characterization of a recombinant l-ribose isomerase from Actinotalea fermentans ATCC 43279.

A putative l-ribose isomerase (EC 5.3.1.

Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli.

Cyanophycin, also known as cyanophycin granule polypeptide (CGP), is a non-ribosomal polypeptide consisting of aspartic acid as a backbone with arginine and lysine as the side chains. CGP has soluble (sCGP) and insoluble (iCGP) forms based on its aqueous solubility. In order to investigate the role of lysine in its physical properties, CGP was prepared with recombinant Escherichia coli cultivated at different temperatures, and the purified sCGP and iCGP were further fractionated with different ethanol concentrations and pHs, respectively.

Identification of substrate-binding and selectivity-related residues of maltooligosyltrehalose synthase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in the synthesis of trehalose. Computer simulations using AutoDock and NAMD were employed to assess the substrate-binding and selectivity-related residues of MTSase. We introduced mutations at residues D411, D610, and R614 to determine the substrate-binding residues of Sulfolobus solfataricus ATCC 35092 MTSase, and introduced mutations at residues P402, A406, and V426 to investigate the enzyme's selectivity-related residues.

pH responsive PEGylation through metal affinity for gene delivery mediated by histidine-grafted polyethylenimine.

Polyethylene glycol (PEG) has been used to enhance the stability of a gene delivery system. The most commonly used approach is to add the PEG molecule by way of chemical conjugation. In this study, we prepared PEG-bearing nitrilotriacetic acid (ntaPEG) followed by chelation with either nickel or zinc ions.

Assessments of growth conditions on the production of cyanophycin by recombinant Escherichia coli strains expressing cyanophycin synthetase gene.

The synthesis of cyanophycin, a biodegradable polymer, is directed by cyanophycin synthetase. Polymerase chain reaction (PCR) cloned the gene cphA coding for cyanophycin synthetase from Synechocystis sp. PCC 6803 into pET-21b followed by transformation into two Escherichia coli hosts.

Characterization of a thermophilic L-rhamnose isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494.

L-Rhamnose isomerase (EC 5.3.1.

Characterization of a thermophilic L-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1.

L-rhamnose isomerase (EC 5.3.1.

Simultaneous mutations up to six distal sites using a phosphorylation-free and ligase-free polymerase chain reaction-based mutagenesis.

In this study, we present an efficient phosphorylation-free and ligase-free PCR-based multiple site-directed mutagenesis that allows simultaneous mutations up to six distal sites. This method could be extended to any plasmid DNA that is isolated from dam(+)Escherichia coli strains, and the results showed that the simultaneously mutagenic efficiencies of quadruple mutation and sextuple mutation were up to 80% and 40%, respectively.

Transcutaneous immunization by lipoplex-patch based DNA vaccines is effective vaccination against Japanese encephalitis virus infection.

Skin, the biggest organ of human body, contains antigen presenting cells such as Langerhans cells (LCs) that modulate various immune responses. The skin therefore is an ideal venue to effect the transcutaneous immunization (TCI). Most current immunization procedures make use of needles and syringes for vaccine administration, which however have raised many safety concerns.

Using trehalose delivered by the intramuscular injection of plasmid DNA as an adjuvant for transgene expression.

Background: Intramuscular injection is a popular and effective approach to administer naked plasmid for transgene expression. The use of an adjuvant can provide a straightforward approach for enhancing transgene expression.

Methods: Expression plasmid was formulated with various concentrations of trehalose for injection into the skeletal muscles of C57BL/6 mice.

Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.

A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method.

In this study, we report a novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method modified from the QuikChange site-directed mutagenesis (QCM). One mutagenic oligonucleotide and one universal flanking primer were used to produce the complementary megaprimers that were then used to amplify the whole plasmid template. This method yields a mutagenesis efficiency ( approximately 90%) similar to that of QCM but uses only one mutagenic oligonucleotide instead of two of them, and the length of the oligonucleotide could be shorter.

Trehalose enhances transgene expression mediated by DNA-PEI complexes.

Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. In this study, we found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides.

Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system.

Background: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors.

Protein engineering of Sulfolobus solfataricus maltooligosyltrehalose synthase to alter its selectivity.

Maltooligosyltrehalose synthase (MTSase) is one of the key enzymes involved in trehalose production from starch and catalyzes an intramolecular transglycosylation reaction by converting the alpha-1,4- to alpha,alpha-1,1-glucosidic linkage. Mutations at residues F206, F207, and F405 were constructed to change the selectivity of the enzyme because the changes in selectivity could reduce the side hydrolysis reaction of releasing glucose and thus increase trehalose production from starch. As compared with wild-type MTSase, F405Y and F405M MTSases had decreased ratios of the initial rate of glucose formation to that of trehalose formation in starch digestion at 75 degrees C when wild-type and mutant MTSases were, respectively, used with isoamylase and maltooligosyltrehalose trehalohydrolase (MTHase).

Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction.

Mutations on aromatic residues of the active site to alter selectivity of the Sulfolobus solfataricus maltooligosyltrehalose synthase.

Mutations Y290F, Y367F, F405Y, and Y409F located near subsite +1 were constructed in maltooligosyltrehalose synthase (MTSase) to alter the selectivity of the enzyme. These mutations were designed to evaluate the effects of hydrophobic interactions and/or hydrogen bondings on transglycosylation and side hydrolysis reactions. The catalytic efficiencies of Y290F MTSase for hydrolysis and transglycosylation reactions were only 6.

Dependence of transgene expression and the relative buffering capacity of dextran-grafted polyethylenimine.

Branched polyethylenimine (PEI) is a cationic polymer capable of forming self-assembly complexes with DNA to become a highly efficient agent used in gene delivery. Conjugation through the primary amines of PEI is a most commonly used approach further to enable the targeting delivery or to improve the stability of the DNA-polymer complexes. An understanding of how the conjugation affects the transfection mechanisms can help in the design of efficient polycationic vectors.

The role of dextran conjugation in transfection mediated by dextran-grafted polyethylenimine.

Background: Conjugation through primary amines is one of the most commonly used methods to modify polycationic vectors for gene delivery. A better understanding of the effect of the conjugation on the mechanisms of transgene expression can help design efficient polycationic vectors.

Methods: Dextran with a molecular weight of 1500 was grafted onto polyethylenimine (PEI) to produce various degrees of grafting in an effort to investigate how the conjugation affected the mechanisms of transgene expression.