RESUMO
Phenylalany--tRNA synthetase is a key enzyme for protein synthesis in Trypanosoma. Its validation as an inhibition. target will enable the development of a new generation of anti-Trypanosoma drugs. However, little is known about the isolation of the Trypanosoma Phenylalanyl-tRNA synthetase. Here we report the cloning, expression, purification, and activity assay of Phenylalanyl-tRNA synthetase from Trypanosoma brucei in Escherichia coli host. We co-cloned the alpha-subunit and beta-subunit of Phenylalanyl-tRNA synthetase from Trypanosoma brucei genomic DNA into the co-expression vector pCOLADuet. We successfully expressed the Trypanosoma brucei Phenylalanyl-tRNA synthetase in E. coli host, purified the whole enzyme by Ni-Hind affinity column and verified it by Western blotting. In addition, we tested its enzymatic activity by isotope labeling. The whole work laid a solid foundation for in vitro the screening and optimization of Trypanosoma brucei phenylalanyl-tRNA synthetase inhibitors.
Assuntos
Clonagem Molecular , Escherichia coli , Genética , Metabolismo , Vetores Genéticos , Genética , Fenilalanina-tRNA Ligase , Genética , Proteínas de Protozoários , Genética , Proteínas Recombinantes , Genética , Metabolismo , Trypanosoma brucei brucei , GenéticaRESUMO
Purpose To clone and express Trypanosoma Leucyl-tRNA synthetase (LeuRS) gene and to complete the purification and activity assay of LeuRS. Methods We cloned the LeuRS gene from T. Brucei genomic DNA,and cloned it into pBS-T and then into the expression vector pET21a( + ) .The expression of T. Brucei LeuRS was carried out in E. coli BL21 (DE3) RIPL host by optimizing the expression conditions. The products are purified by His-Bind affinity column and verified by Western blot . Enzymatic activity was detected by isotope labeling. Results A DNA fragment at length of 3.2 kb was amplified. Both restriction analysis and sequencing analysis proved that recombinant plasmid pET21a( + )/LeuRS was correctly constructed. The expressed product contained about 20% of total somatic soluble protein and reached a purity of 85% after purification . It was verified by the Western blot. Enzyme unit per millilitre of purified products was about 72. Conclusion Here we report the successful clone, expression and purification of LeuRS from Trypanosoma Brucei ( T. Brucei) in bacterial host. In addition, we tested its enzymatic activity by isotope labeling.This work would be helpful to the design and in vitro screening of Trypanosoma LeuRS inhibitors.
RESUMO
AIM:To establish a method of determining the content of chlorogenic acid,paracetmol,vitamin C and chlorphenamine maleate in Vitamin C Yinqiao Tablets. METHODS: The separation was performed in the C_(18) colunm with the mobile phase of methanol-acetonitril-0.02% phosphate acid(5∶10∶85).The flow rate was(1.0 mL/min) with the wavelength at 219 nm and 310 nm,the temperature of column was 35 ℃. RESULTS: The calibration curves were linear in the ranges of 0.03-0.13 ?g for chlorogenic acid,0.94-4.68 ?g for paracetmol,0.49-2.44 ?g for Vitamin C,0.01-0.05 ?g for chlorphenamine maleate,the average recoveries were not less than 98%,respectively. CONCLUSION: The method is simple,rapid and with satisfactory results.It is suitable for quality control of Vitamin C Yinqiao Tablets.
RESUMO
AIM:To establish the method of determining notoginsenoside R_1,ginsenoside Rg_1 and Rb_1 in Xinning Tablet(Radix et Rhizoma Salvae Miltiorrhizae,Radix et Rhizoma notoginseng,Flos Carthami,Rhizoma Chuanxiong,ect).METHODS:HPLC-ELSD was used to determine notoginsenoside R_1,ginsenoside Rg_1 and Rb_1 in Xinning Tablet.The separatrion was performed on C_ 18 colunm with acetonitrile and water being used as a gradient program at 35 ℃.The elution program was(0-5 min,20%-25% acetonitrile;5-20 min,25%-45% acetonitrile),drift tube temperature was at 70 ℃,gas flow rate of 2.0 L/min.RESULTS:3 saponins were separated well.Average recoveries were 102.32% for notoginsenoside R_1 100.73% for ginsenoside Rg_1;101.40% for ginsenoside Rb_1,respectively.CONCLUSION:The method is simple and rapid and with satisfactory results and is suitable for quality control of Xinning Tablet.