Tsuei-Yun Fang

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. This method can be further extended to double mutations that are located at distant sites by using two mutagenic oligonucleotides and even to site saturation mutagenesis by introducing randomized codons.

Characterization of a Thermophilic L-Rhamnose Isomerase from Thermoanaerobacterium saccharolyticum NTOU1

L-rhamnose isomerase (EC 5.3.1.14, L-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-Rhi gene encoding L-Rhi was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.

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. The culture conditions for cyanophycin production were investigated, and the molecular weight and compositions of purified cyanophycin were analyzed. The results showed that E. coli BL21‐CodonPlus(DE3)‐RIL could produce 120 mg cyanophycin per gram dry cell weight in terrific medium. The purified cyanophycin consisted of insoluble and soluble forms at pH 7. The insoluble form had a higher molecular weight (20–32 kDa) than the soluble form (14–25 kDa). Both forms are composed of three major amino acids, aspartic acid, arginine, and lysine, and the insoluble form showed a higher arginine/lysine molar ratio (4.61 ± 0.31) than the soluble form (0.89 ± 0.05). In addition, the nitrogen sources could affect the yields of insoluble and soluble forms of cyanophycin. The medium containing additional lysine could enhance the proportion of the soluble form, but had little effect on the lysine and arginine percentages of both soluble and insoluble forms. The medium containing additional arginine slightly decreased the proportion of soluble form and altered its amino acid composition, with a minimal effect on the lysine and arginine percentages in the insoluble form.

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

L-Rhamnose isomerase (EC 5.3.1.14, l-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-rhi gene encoding L-RhI was PCR-cloned from Caldicellulosiruptor saccharolyticus ATCC 43494 and then expressed in Escherichia coli. A high yield of active L-RhI, 3010 U/g of wet cells, was obtained after 20 °C induction for 20 h. The enzyme was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 90 °C. The enzyme was stable at pH values ranging from 4 to 11 and retained >90% activity after a 6 h incubation at 80 °C and pH 7-8. Compared with other previously characterized L-RhIs, the L-RhI from C. saccharolyticus ATCC 43494 has a good thermostability, the widest pH-stable range, and the highest catalytic efficiencies (k(cat)/K(M)) against L-rhamnose, L-lyxose, L-mannose, D-allose, and D-ribose, suggesting that this enzyme has the potential to be applied in rare sugar production.

Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.

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.

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.

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. In an effort to explore the mechanisms, we examined how the timing of trehalose treatments and the durations of trehalose affected the percentages of cells expressing green fluorescent protein and the levels of intracellular ethidium monoazide labeled plasmid. Treatments with trehalose for 5–120 min prior to transfection could cause drops in transfection efficiency by 30–50%; such treatments, however, hardly affected the amounts of intracellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA‐PEI complexes. Additionlly, the extent of enhancement in transgene expression strongly depended on the duration of trehalose. As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA‐PEI complexes possibly by affecting the mechanisms of intracellular trafficking.

Characterization of a Recombinant L-Ribose Isomerase From Geodermatophilus Obscurus Dsm 43160 And Application of this Enzyme to the Production of L-Ribose from L-Arabinose

L-Ribose isomerase (L-RI) catalyzes the aldose-ketose isomerization between L-ribose and L-ribulose. In this study, a putative L-RI gene of Geodermatophilus obscurus DSM 43160 was cloned by PCR into pET-15b and pET-21b, respectively. The cloned target gene was expressed in Escherichia coli. The recombinant N-His-tagged and C-His-tagged proteins exhibited L-RI activity. Both N- and C-His-tagged L-RIs were purified from cell-free extracts by metal-affinity and ion-exchange chromatography. The purified N-His-tagged L-RI demonstrated its optimal activity at 30-40 oC and pH of 9 (in glycine-NaOH buffer). The enzyme was stable at pH 7-9 and more than 90% activity was retained after incubation at 40℃ for 2 h. Metal ions were not required for N-His-tagged L-RI activity to occur, but Hg2+ inhibited its activity completely. The conversion rates of L-arabinose to L-ribose by combining Thermoanaerobacterium saccharolyticum NTOU1 L-arabinose isomerase and G. obscurus DSM43160 N-His-tagged L-RI at 30 oC and 40 oC were 15.9% and 12.5% (mol mol-1), respectively. Results obtained from this study suggest a potential application of this recombinant L-RI for the L-ribose production from L-arabinose.

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. Polyethylenimine was grafted with histidine (hisPEI) and used as a primary gene carrier to form complexes with DNA. PEGylation was performed by incubating the complexes with chelated ntaPEG. It was noted that the coating of the chelated ntaPEG could provide a shielding effect against aggregation induced by bovine serum albumin and DNA release induced by heparin displacement, respectively. The coating was also found to improve cellular viability and maintain the transfection efficiency at a moderate level. The coated ntaPEG could dissociate from the complexes in an acidic condition of pH 4, suggesting that dePEGylation might occur in some acidic intracellular organelles, such as endosomes. This simple and effective PEGylation approach could be extended to other delivery systems to enhance the stability and to facilitate the dePEGylation process.