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Zhang Y.,University of Sichuan | Zhang Y.,Sichuan Industrial Institute of Antibiotics | Wang L.,University of Sichuan | Zhang H.,University of Sichuan | And 2 more authors.
Chromatographia | Year: 2011

A simple, specific and sensitive RP-LC method was developed and validated for the determination of tetrandrine in rat whole blood for the first time. Chromatographic separation was performed on a Welchrom™ C18 analytical column at a flow rate of 1.0 mL min-1, using a mixture of methanol-water containing 0.6% triethylamine and 0.16% phosphoric acid as mobile phase. The wavelength for UV detection was set at 225 nm. Sample preparation involved a liquid-liquid extraction using n-hexane. The calibration curve was linear with r 2 > 0.99 over a concentration range of 0.03-6.4 μg mL-1 in rat whole blood with a lower limit of quantification of 0.03 μg mL-1. The intra- and inter-day precisions were 1.33-4.55 and 3.33-4.65%, respectively, and the intra- and inter-day accuracy ranged from -5.24 to 0.90% and -1.05 to 0.63%, respectively. No endogenous compounds were found to interfere with the analytes. Tetrandrine was stable for 8 h at room temperature, 24 h at 4°C in rat whole blood, and for 30 days at -20°C after being prepared. For the first time, the present method was successfully applied to the pharmacokinetic studies of tetrandrine in rats after intravenous administration of three different doses. The results indicated that the pharmacokinetics of tetrandrine on rats was a first-order process. © 2011 Springer-Verlag.

Qu F.,Sichuan Industrial Institute of Antibiotics
Wei sheng wu xue bao = Acta microbiologica Sinica | Year: 2012

The intracellular alpha-amino acid ester hydrolase (AEH) from Xanthomonas rubrillineans was purified and characterized. AEH was extracted by butyl acetate, and then purified to electrophoretic homogeneity by calcium phosphate gel precipitation, ammonium sulfate fraction precipitation, anion exchange with DEAE Sephadex A-50, cation exchange with CM cellulose 52, and Sephadex G 200 column chromatography. The subunit molecular mass of AEH was 70 kDa by SDS-PAGE. The optimal reaction pH for cefaclor synthesis was 6.8, and optimal temperature was 42 degrees C. The enzyme was stable between pH 5.0 and 8.0, and at 35 degrees C. The enzyme activity was enhanced by Mn2+ and Ca2+, however was strongly inhabited by Cu2+, Fe2+ and high concentration of acetone. The kinetic parameters that the enzyme hydrolyzed D-Phenylglycine methyl ester, D-Hydroxyphenylglycine methyl ester and cefaclor were determined, and the values of k(cat)/K(m) were 123.7 +/-3.7, 2.9 +/- 0.6 and 101.3 +/- 2.1 mmol(-1) x s(-1) x L respectively. The k(cat)/K(m) values indicated that the enzyme hydrolyzed D-Phenylglycine methyl ester more efficiently than other substrates. The mechanism of enzymatic reaction with bi-substrates by AEH is Ping-Pong kinetics, and the k(cat) value that the enzyme catalyzed the synthesis of cefaclor is 547.3 +/- 38.2 s(-1). The studies of AEH from Xanthomonas rubrillineans were rare, and our research may provide an important basis for industrial application of AEH for beta-lactam antibiotics synthesis.

Fu X.-J.,Sichuan Industrial Institute of Antibiotics | Zhang Y.-X.,Chengdu Institute of Biological Products Co. | Dai Y.-J.,Chengdu Institute of Biological Products Co. | Wang M.-R.,Chengdu Institute of Biological Products Co.
Yaoxue Xuebao | Year: 2013

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1 000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTMl-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol·L-1) was selected from the humanized library of 1012 members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.

He M.,Babraham Institute | He Y.,Sichuan Industrial Institute of Antibiotics | Luo Q.,University of Sichuan | Wang M.,Sichuan Industrial Institute of Antibiotics
Process Biochemistry | Year: 2011

Proteomics and biotechnology studies require simple and rapid methods to convert the genetic information into proteins. Whereas heterologous protein expression in living cells is a time-consuming process, in vitro translation directs protein synthesis in hours from added linear PCR DNA without the need for E. coli cloning, thus providing an attractive alternative to cell-based methods for high throughput production of proteins. Moreover, the open nature of cell-free systems and availability of various prokaryotic and eukaryotic cellular lysates offers a flexible choice of conditions for synthesis of folded proteins or production of "difficult" proteins that are not possible by in vivo systems. Finally, cell-free extracts express protein populations in a single reaction, allowing for the development of powerful proteomic tools. This article will review the recent advances in cell-free protein expression and its applications in biotechnology, proteomics and fundamental biological research. © 2010 Elsevier Ltd.

Chen F.,Chinese Academy of Sciences | Chen F.,Sichuan Industrial Institute of Antibiotics | Lin L.,Chinese Academy of Sciences | Lin L.,Northeast Agricultural University | And 7 more authors.
Applied Microbiology and Biotechnology | Year: 2011

To investigate the distribution of dTDP-glucose-4,6-dehydratase (dTGD) gene and diversity of the potential 6-deoxyhexose (6DOH) glycosylated compounds in marine microorganisms, a total of 91 marine sediment-derived bacteria, representing 48 operational taxonomic units and belonging to 25 genera, were screened by polymerase chain reaction. In total, 84% of the strains were dTGD gene positive, suggesting 6DOH biosynthetic pathway is widespread in these marine sediment-derived bacteria. BLASTp results of dTGD gene fragments indicate a high chemical diversity of the potential 6DOH glycosylated compounds. Close phylogenetic relationship occurred between dTGDs involved in the production of same or similar 6DOH glycosylated compounds, suggesting dTGD can be used to predict the structure of potential 6DOH glycosylated compounds produced by new strains. In two cases, where dTGD shared ≥85% amino acid identity and close phylogenetic relationship with their counterparts, 6DOH glycosylated compounds were accurately predicted. Our results demonstrate that phylogenetic analysis of dTGD gene is useful for structure prediction of glycosylated compounds from newly isolated strains and can therefore guide the chemical purification and structure identification process. The rapid identification of strains that possess dTGD gene provides a bioinformatics assessment of the greatest potential to produce glycosylated compounds despite the absence of fully biosynthetic pathways or genome sequences. © 2011 Springer-Verlag.

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