A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restr...A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no expressed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the expression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.展开更多
DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeas...DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. These fragments were inserted into a site between the AOX 1-promoter and -terminator in pPICZ A to be expressed by P. pastoris. The expression vector was linearized, and introduced into P. pastoris GS115 by electroporation. After the checking of several transformants with PCR to ensure a precise insertion, one was selected and cultured to examine antibody production. The level of production reached 10 mg/L in a flask with medium containing 1% methanol. The heavy chain and light chain of the product were assembled to form a hetero tetramer, as detected by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal amino acid sequencing revealed that the signal peptides of both chains were well processed. The mobility of the product in SDS-PAGE after treatment with Peptide N-Glycosidase F indicated the heavy chain to be N-glycosylated. Further analysis of the N-glycans with a mass spectrometer revealed a mixture of Man9-GlcNAc2, Man10-GlcNAc2, Man11-GlcNAc2 and Man12-GlcNAc2, but no hyper-mannosylated glycans. ELISA, surface plasmon resonance, and flow cytometric studies showed the affinity curve and Kd value for the antigen, HER II, and reactivity to a HER2-overexpressing breast cancer cell-line, SK-BR-3, to be almost the same as for the clinically used trastuzumab produced by CHO.展开更多
This study aimed at exploring for new natural peptides with strong inhibitory capabilities on α-amylase, the main metabolic enzyme that regulates mellitus diabetes, in order to contribute in controlling this global p...This study aimed at exploring for new natural peptides with strong inhibitory capabilities on α-amylase, the main metabolic enzyme that regulates mellitus diabetes, in order to contribute in controlling this global pandemic. It has consisted in heat shock (to 60°C, 70°C, 80°C, 90°C and 100°C for 10, 20 and 30 minutes) of crude proteins extracted from biomass and extracellular parts of Saccharomyces cerevisiae under cultivation, and from the digestive fluid of the giant snail Achatina achatina, and in-vitro assays of the resulting solutions, as effectors, in human α-amylase catalyzing reactions. The results showed that whatever the temperature and time of treatment, an increase (from 2.65 to 3.98-fold) in proteins concentration was noticed. When blended up to 75 microliters in reaction mixtures, the three peptide extracts showed beyond 11% of inhibition of initial α-amylase activity. By reducing samples volume, only 5 microliters of the studied peptide extracts representing 4.70 μg of S. cerevisiae biomass peptides, 0.55 μg of S. cerevisiae extracellular peptides or 1.05 μg of peptides from the digestive fluid A. achatina were quite sufficient to induce complete (100%) inhibition of the human α-amylase activity. Compared to the inhibitory effect obtained from 2.50 μg of acarbose, a renowned antidiabetic, the studied peptide effectors showed more pronounced inhibitory activities. So, we can positively state that S. cerevisiae as well as A. achatina are both capable of synthesizing proteins made up of small inhibitory peptides which deserve purification and structural analysis for potential exploitation as healthy antidiabetic drugs.展开更多
Carrot （Daucus carom） is a valuable source of health promoting ingredients such as anthocyanin, carotenes, phenolic compounds etc. These substances are important to man as a source of pharmaceuticals, fragrance, agr...Carrot （Daucus carom） is a valuable source of health promoting ingredients such as anthocyanin, carotenes, phenolic compounds etc. These substances are important to man as a source of pharmaceuticals, fragrance, agrochemicals as well as food additives and used for prevention of many chronic diseases. Since these activities may be correlated with the presence of antioxidant compounds, extract of carrot and carrot callus were evaluated for their anthocyanin, flavonoids and total phenolic content as well as total antioxidant activity. Anthocyanin content was measured by spectrophotometric method. Total phenols and flavonoids were analyzed according to the Folin-Ciocalteu method and total antioxidant activity was assessed by ferric reducing/antioxidant power （FRAP） assay. Anthocyanin, flavonoids and total phenolic content were estimated to be 9.36 mg%, 46.96 mg% and 57.01 mg% for callus and 6.82 mg%, 32.96 mg% and 42.69 mg% for carrot, respectively, on fresh weight basis. The total antioxidant activity for the callus and carrot was found to be 51.13 mg, 118.77 mg, 91.08 mg and 140.08 mg equivalent and 79.40 mg, 184.44 mg, 141.43 mg and 217.52 mg equivalent to gallic acid, vitamin C, butylated hydroxyanisole （BHA） and trolox, respectively, when expressed per 100 g on fresh weight basis. The antioxidant activity of fresh carrot was found to be higher compared to its callus.展开更多
文摘A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no expressed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the expression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.
文摘DNA fragments encoding the light chain and heavy chain genes of an anti-human HER II antibody, trastuzumab, fused with an egg-lysozyme signal peptide were synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. These fragments were inserted into a site between the AOX 1-promoter and -terminator in pPICZ A to be expressed by P. pastoris. The expression vector was linearized, and introduced into P. pastoris GS115 by electroporation. After the checking of several transformants with PCR to ensure a precise insertion, one was selected and cultured to examine antibody production. The level of production reached 10 mg/L in a flask with medium containing 1% methanol. The heavy chain and light chain of the product were assembled to form a hetero tetramer, as detected by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal amino acid sequencing revealed that the signal peptides of both chains were well processed. The mobility of the product in SDS-PAGE after treatment with Peptide N-Glycosidase F indicated the heavy chain to be N-glycosylated. Further analysis of the N-glycans with a mass spectrometer revealed a mixture of Man9-GlcNAc2, Man10-GlcNAc2, Man11-GlcNAc2 and Man12-GlcNAc2, but no hyper-mannosylated glycans. ELISA, surface plasmon resonance, and flow cytometric studies showed the affinity curve and Kd value for the antigen, HER II, and reactivity to a HER2-overexpressing breast cancer cell-line, SK-BR-3, to be almost the same as for the clinically used trastuzumab produced by CHO.
文摘This study aimed at exploring for new natural peptides with strong inhibitory capabilities on α-amylase, the main metabolic enzyme that regulates mellitus diabetes, in order to contribute in controlling this global pandemic. It has consisted in heat shock (to 60°C, 70°C, 80°C, 90°C and 100°C for 10, 20 and 30 minutes) of crude proteins extracted from biomass and extracellular parts of Saccharomyces cerevisiae under cultivation, and from the digestive fluid of the giant snail Achatina achatina, and in-vitro assays of the resulting solutions, as effectors, in human α-amylase catalyzing reactions. The results showed that whatever the temperature and time of treatment, an increase (from 2.65 to 3.98-fold) in proteins concentration was noticed. When blended up to 75 microliters in reaction mixtures, the three peptide extracts showed beyond 11% of inhibition of initial α-amylase activity. By reducing samples volume, only 5 microliters of the studied peptide extracts representing 4.70 μg of S. cerevisiae biomass peptides, 0.55 μg of S. cerevisiae extracellular peptides or 1.05 μg of peptides from the digestive fluid A. achatina were quite sufficient to induce complete (100%) inhibition of the human α-amylase activity. Compared to the inhibitory effect obtained from 2.50 μg of acarbose, a renowned antidiabetic, the studied peptide effectors showed more pronounced inhibitory activities. So, we can positively state that S. cerevisiae as well as A. achatina are both capable of synthesizing proteins made up of small inhibitory peptides which deserve purification and structural analysis for potential exploitation as healthy antidiabetic drugs.
文摘Carrot （Daucus carom） is a valuable source of health promoting ingredients such as anthocyanin, carotenes, phenolic compounds etc. These substances are important to man as a source of pharmaceuticals, fragrance, agrochemicals as well as food additives and used for prevention of many chronic diseases. Since these activities may be correlated with the presence of antioxidant compounds, extract of carrot and carrot callus were evaluated for their anthocyanin, flavonoids and total phenolic content as well as total antioxidant activity. Anthocyanin content was measured by spectrophotometric method. Total phenols and flavonoids were analyzed according to the Folin-Ciocalteu method and total antioxidant activity was assessed by ferric reducing/antioxidant power （FRAP） assay. Anthocyanin, flavonoids and total phenolic content were estimated to be 9.36 mg%, 46.96 mg% and 57.01 mg% for callus and 6.82 mg%, 32.96 mg% and 42.69 mg% for carrot, respectively, on fresh weight basis. The total antioxidant activity for the callus and carrot was found to be 51.13 mg, 118.77 mg, 91.08 mg and 140.08 mg equivalent and 79.40 mg, 184.44 mg, 141.43 mg and 217.52 mg equivalent to gallic acid, vitamin C, butylated hydroxyanisole （BHA） and trolox, respectively, when expressed per 100 g on fresh weight basis. The antioxidant activity of fresh carrot was found to be higher compared to its callus.
