Ke-Di Cheng

Peking Union Medical College Hospital, Peping, Beijing, China

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Publications (35)30.25 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
    Yao xue xue bao = Acta pharmaceutica Sinica 05/2015; 50(5):627-32.
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    ABSTRACT: Three cyclotides were isolated from the whole plant of Viola yedoensis in this study. The two, vary peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately. The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again. Besides, the IC50 value of cycloviolacin VYI against influenza A H1N1 virus was (2.27 +/- 0.20) microg x mL(-1). It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.
    Yao xue xue bao = Acta pharmaceutica Sinica 06/2014; 49(6):905-12.
  • Zhi-Biao Wang · Xi Chen · Wei Wang · Ke-Di Cheng · Jian-Qiang Kong ·
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    ABSTRACT: OSW-1 is a promising antitumor glycoside present in the Ornithogalum saundersiae plant. Biosynthesis of the p-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically by phenylpropanoid biosynthetic pathway, but molecular biological characterization of related genes has been insufficient. Phenylalanine ammonia lyase (PAL, EC, which catalyzes the deamination of L-phenylalanine to yield trans-cinnamic acid, plays a key role in phenylpropanoid metabolism. Thus, the study on the characterization of the genes involved in the OSW-1 biosynthetic pathway, particularly the well-documented genes such as PAL, is essential to further the understanding of the biosynthesis of OSW-1. Here, transcriptomic sequencing of O. saundersiae was performed to speed up the identification of a large number of genes related to OSW-1 biosynthesis. De novo assembly of the transcriptome sequence provided 210733 contigs, 104, 180 unigenes, and four unigenes showing high similarities with PALs. Two full-length cDNAs encoding PALs (OsaPAL2 and OsaPAL62) from O. saundersiae were cloned using sequence information from these four unigenes. The PAL and tyrosine ammonia lyase (TAL) activities of recombinant OsaPAL proteins were unambiguously determined by HPLC with UV and MS detection as well as by NMR spectroscopy. Subsequently, a series of site-directed mutants were generated with the aim of improving enzyme activity and investigating the importance of particular residues in determining substrate selectivity. The results reveal that the Phe-to-His mutants, i.e., OsaPAL2F134H and OsaPAL62F128H, exhibited higher TAL activity than the corresponding wild types, providing direct evidence that the Phe residue is responsible for substrate specificity. Mutagenesis studies also demonstrated that the Thr-to-Ser mutants, i.e., OsaPAL2T196S and OsaPAL62T194S, showed significantly higher substrate affinity than the wild types. Furthermore, the Gly-to-Ala mutants, i.e., OsaPAL2G209A and OsaPAL62G207A, showed higher PAL and TAL activities. These findings provide further insight into the genes responsible for OSW-1 biosynthesis and will facilitate the future application of OsaPALs in synthetic biology.
    RSC Advances 01/2014; 4(52):27159. DOI:10.1039/c4ra03385j · 3.84 Impact Factor
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    ABSTRACT: Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produces significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bio-conversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. Combination of the de novo sequencing of β-xylosidase isolated from Lentinula edodes with the RT-PCR/RACE strategy, we cloned two cDNA variants Lxyl-p1-1 and Lxyl-p1-2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-D-xylosidase/β-D-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized and their 3.6 kb genomic DNAs (G-Lxyl-p1-1, G-Lxyl-p1-2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, catalytic nucleophile, catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km up to 1.07/s/mM against 7-β-xylosyl-10-deacetyltaxol. Importantly, at the substrate concentrations up to 10 mg/ml (over-saturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with over 85% conversion rate and the highest yield of 8.42 mg/ml within 24 h, which is much higher than those of previous reports. Therefore, our discovery may bring significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work may also lead to further studies on detailed mechanisms of how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.
    Molecular &amp Cellular Proteomics 05/2013; 12(8). DOI:10.1074/mcp.M113.030619 · 6.56 Impact Factor
  • Jian-Qiang Kong · Wei Wang · Ke-Di Cheng · Ping Zhu ·
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    ABSTRACT: The first-line drug artemisinin is widely used against malaria. Commercially available artemisinin is extracted from plants. However, the lack of sufficient raw material, artemisinin and the cost associated with the drug's manufacture have limited the supply of ACT to most malaria sufferers in the Developing World. As such, it is important to develop a low cost, fine to environment and high-quality method to supply sufficient and reliable quantities of artemisinin in the future. The field of synthetic biology, which utilizes cell factories to manipulate microbial metabolism to enhance the production of artemisinin and its intermediates, has a particularly strong impact by providing new platforms for chemical production. After a brief introduction of the artemisinin biosynthetic pathway, the present review focuses on the introduction of artemisinin biosynthetic genes, such as the genes encoding amorpha-4, 11-diene monooxygenase, NADPH: cytochrome P450 oxidoreductase, artemisinic aldehyde delta 11(13) reductase and aldehyde dehydrogenase. The review also addresses general considerations for potential contributions of synthetic biology to artemisinin production, with an emphasis on factors influencing interest compounds production in chassis cells.
    Yao xue xue bao = Acta pharmaceutica Sinica 02/2013; 48(2):193-205.
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    ABSTRACT: The synthetic biology matures to promote the heterologous biosynthesis of the well-known drug paclitaxel that is one of the most important and active chemotherapeutic agents for the first-line clinical treatment of cancer. This review focuses on the construction and regulation of the biosynthetic pathway of paclitaxel intermediates in both Escherichia coli and Saccharomyces cerevisiae. In particular, the review also features the early efforts to design and overproduce taxadiene and the bottleneck of scale fermentation for producing the intermediates.
    Yao xue xue bao = Acta pharmaceutica Sinica 02/2013; 48(2):187-92.
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    ABSTRACT: Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.
    Yao xue xue bao = Acta pharmaceutica Sinica 02/2013; 48(2):228-35.
  • Chang-Qing Li · Wen-Chao Liu · Ping Zhu · Jin-Ling Yang · Ke-Di Cheng ·
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    ABSTRACT: Several molecular techniques were employed to document the bacterial diversity associated with the marine sponge Gelliodes carnosa. Cultivation-dependent and cultivation-independent methods were used to obtain the 16S rRNA gene sequences of the bacteria. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the bacterial community structure was highly diverse with representatives of the high G + C Gram-positive bacteria, cyanobacteria, low G + C Gram-positive bacteria, and proteobacteria (α-, β-, and γ-), most of which were also found in other marine environments, including in association with other sponges. Overall, 300 bacterial isolates were cultivated, and a total of 62 operational taxonomic units (OTUs) were identified from these isolates by restriction fragment length polymorphism (RFLP) analysis and DNA sequencing of the 16S rRNA genes. Approximately 1,000 16S rRNA gene clones were obtained by the cultivation-independent method. A total of 310 clones were randomly selected for RFLP analysis, from which 33 OTUs were acquired by further DNA sequencing and chimera checking. A total of 12 cultured OTUs (19.4% of the total cultured OTUs) and 13 uncultured OTUs (39.4% of the total uncultured OTUs) had low sequence identity (≤97%) with their closest matches in GenBank and were probably new species. Our data provide strong evidence for the presence of a diverse variety of unidentified bacteria in the marine sponge G. carnosa. A relatively high proportion of the isolates exhibited antimicrobial activity, and the deferred antagonism assay showed that over half of the active isolates exhibited a much stronger bioactivity when grown on medium containing seawater. In addition to demonstrating that the sponge-associated bacteria could be a rich source of new biologically active natural products, the results may have ecological implications. This study expands our knowledge of the diversity of sponge-associated bacteria and contributes to the growing database of the bacterial communities within sponges.
    Microbial Ecology 07/2011; 62(4):800-12. DOI:10.1007/s00248-011-9896-6 · 3.12 Impact Factor
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    ABSTRACT: Human enterovirus 71 (EV71) is one of the major etiological agents for the hand, foot, and month disease (HFMD) and is causing frequent, widespread occurrence in the mainland of China. The single positive-stranded RNA genome of EV71 is translated into a single polyprotein which is autocleavaged into structural and nonstructural proteins. The functions of many nonstructural proteins characterized in the life cycle of virus are potential targets for blocking viral replication. This article reviews the studies of the structures and functions of nonstructural proteins of EV71 and the anti-enterovirus 71 drugs targeting on these nonstructural proteins.
    Yao xue xue bao = Acta pharmaceutica Sinica 07/2011; 46(7):753-61.
  • Rui-Yu Zhao · Wei Xiao · Hai-Li Cheng · Ping Zhu · Ke-Di Cheng ·
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    ABSTRACT: The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5'-untranslated region (UTR), 3'-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS-PAGE and western blot. GC-MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.
    Journal of Industrial Microbiology 11/2010; 37(11):1171-82. DOI:10.1007/s10295-010-0764-z · 2.44 Impact Factor
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    ABSTRACT: NADPH-cytochrome P450 reductase (CPR), a partner for P450 monooxygenases, serves as the electron donor to almost all eukaryotic cytochrome P450s. One cDNA (TchCPR) encoding cytochrome P450 reductase of T. chinensis was isolated from callus cells. The cDNA contains an open reading frame of 2154 nucleotides which encodes a protein of 717 amino acid residues. The TchCPR has higher similarity to other CPRs of gumnosperms (>82%) than that of angiosperms (<74%). The recombinant full-length TchCPR and a series of N-terminal truncated constructs with N-terminal fusion of His Tag were obtained and induced to express in E. coli B121(DE3), and then purified using affinity chromatography. The truncated forms of N-terminal more than 61 amino acid residues could be efficiently expressed while the truncated mutant of N-terminal 48 amino acid residues and the wild-type TchCPR were not successfully expressed in E. coli cells. The activity of the truncated TchCPR was assayed by measuring the reduction of cytochrome C. The electron transfer activity of the recombinantly purified CPRT61 was 1.6057 nmol of cytochrome C reduced per min per microg TchCPR reductase, and it is higher than that of the other four truncated forms.
    Hereditas (Beijing) 11/2010; 32(11):1187-94.
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    ABSTRACT: The cyclotides are a family of cyclic "mini" proteins that occur in Violaceae, Rubiaceae and Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three disulfide bonds. To study the natural cyclotides of V tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately. The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol. The subfractions were directly reduced by DTT and analyzed by reverse-phase HPLC. The peaks with different retention times were shown on the profile of RP-HPLC and collected. The cyclotides were speculated based on masses range from 3 000 to 3 500 Da. The purified cyclotides were reduced with DTT, alkylated with iodoacetamide, and then were cleaved with endoproteinase Glu-C, endoproteinase Lys-C and Trypsin, separately. The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer. A new cyclotide, cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system. So the method for the isolation and characterization of cyclotides was quickly built up in succession.
    Yao xue xue bao = Acta pharmaceutica Sinica 11/2010; 45(11):1402-9.
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    ABSTRACT: Influenza A/H1N1 virus-encoded nonstructural, or NS1, protein inhibits the 3'-end processing of cellular pre-mRNAs by binding the cellular protein: the 30-kDa subunit of CPSF (cleavage and polyadenylation specificity factor, CPSF30). CPSF30 binding site of the NS1 protein is a potential target for the development of drugs against influenza A/H1N1 virus. A yeast two-hybrid screening system was constructed and used for screening Chinese medicines that inhibit the interaction of the A/H1N1 flu NS1 protein and human CPSF30 protein. The NS1 gene of A/H1N1 virus was amplified by consecutive polymerase chain reaction (PCR), and the human CPSF30 gene of HeLa cell cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Then the two gene fragments confirmed by sequencing were subcloned into the yeast expression vectors pGBKT7 and pGADT7, respectively. The two constructs, bait vector pGBKNS1 and prey vector pGADCPSF, were co-transformed into yeast AH109. The eight individual yeast colonies were picked and subjected to verification by PCR/gel electrophoresis. The inhibition of the NS1-CPSF30 interaction was allowed the identification of selective inhibitors. The four of more than thirty identified Chinese medicines, including 'Shuanghuanglian oral liquid', showed the strong inhibition of the NS1-CPSF30 interaction.
    Yao xue xue bao = Acta pharmaceutica Sinica 03/2010; 45(3):388-94.
  • Jian-Qiang Kong · Yong Huang · Jun-Hao Shen · Wei Wang · Ke-Di Cheng · Ping Zhu ·
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    ABSTRACT: Amorpha-4,11-diene synthase (ADS) can convert farnesyl pyrophosphate (FPP) to amorpha-4, 11-diene, a precursor of artemisinin. ADS plays an important role in the biosynthesis of artemisinin. This review summarizes the molecular biology and metabolic engineering study of ADS in recent years. The genomic DNA and its cDNA sequences of amorpha-4, 11-diene synthase were cloned from Artemisia annua L. The cDNA encoding amorpha-4, 11-diene synthase contains a 1 641 bp open reading frame coding for 546 amino acids. ADS shows a broad pH optimum and an absolute requirement for divalent metal ions as cofactors. The specificity of ADS to the substrates and products is not high and the formation of amorpha-4, 11-diene by ADS from FPP is achieved by an initial 1, 6-closure with subsequent 1, 10-closure. The ADS cDNA cloned from Artemisia annua L, or totally synthesized by PCR, was introduced into different hosts including E. coli, S. cerevisiae, Nicotiana tabacum L. Arabidopsis thaliana and A. nidulans resulting in varied engineering microorganisms and cells producing amorpha-4, 11-diene. The way to improve the production of amorpha-4, 11-diene was investigated by two strategies such as improving the supply of substrate and directing FPP flux to amorpha-4, 11-diene production from competing pathways.
    Yao xue xue bao = Acta pharmaceutica Sinica 12/2009; 44(12):1320-7.
  • Jian-qiang Kong · Jun-hao Shen · Yong Huang · Wei Wang · Ke-di Cheng · Ping Zhu ·
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    ABSTRACT: Plasmid-carrying Saccharomyces cerevisia (W303-1B[pYeDP60/G/ADS]) and genome-transformed S. cerevisia (W303-1B[rDNA:ADS]), both harboring amorpha-4,11-diene synthase (ADS) gene were constructed to investigate the production of amorpha-4,11-diene. The recombinant plasmid pYeDP60/G/ADS that harbors the ADS gene was transformed into S. cerevisiae W303-1B, resulting in the engineered yeast W303-1B[pYeDP60/G/ADS], which contains multi-copies of the plasmid. The ADS gene expression cassette was obtained by PCR amplification of the pYeDP60/G/ADS template, and then introduced into S. cerevisiae W303-1B to obtain the engineered yeast W303-1B[rDNA:ADS], in which the ADS gene was integrated into the rDNA locus of the yeast genome through the homologous recombination. GC-MS analysis confirmed that both of the engineered yeasts could produce amorpha-4,11-diene. Moreover, the amorpha-4,11-diene yield of W303-1B[pYeDP60/G/ADS] was higher than that of W303-1B[rDNA:ADS]. Southern blot analysis showed that there is only one copy of ADS gene in the genome of W303-1B[rDNA:ADS]. It implied that the amorpha-4,11-diene yield can be improved by increasing the ADS gene copies.
    Yao xue xue bao = Acta pharmaceutica Sinica 11/2009; 44(11):1297-303.
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    ABSTRACT: BmK AngM1 is an analgesic peptide from the venom of Buthus martensii Karsch (BmK). The synthetic gene encoding BmK AngM1 was optimized on the basis of its cDNA sequence and the codon usage preference of Pichia pastoris. The codon-optimized gene was cloned into pPIC9K and then transformed into P. pastoris. SDS-PAGE and Western blot analysis showed that the recombinant BmK AngM1 (rBmK AngM1) was expressed by the addition of methanol to the medium, and its maximum production reached above 500mg/l. The purified rBmK AngM1 could be obtained efficiently by Nickel affinity chromatography. Analgesic bioassay, by the mouse-twisting model, showed that rBmK AngM1 had evident analgesic effect with an ED50 of 0.5mg/kg.
    World Journal of Microbiology and Biotechnology 11/2009; 25(11):2053-2056. DOI:10.1007/s11274-009-0108-1 · 1.78 Impact Factor
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    ABSTRACT: Dipeptidyl peptidase (DPP) IV inhibitors provide a new strategy for the treatment of type 2 diabetes. Human DPP-IV gene was cloned from differentiated Caco-2 cells and expressed in Pichia pastoris. The recombinant enzyme was used in a new system for screening of DPP-IV inhibitors. By high throughput screening, a novel compound (W5188) was identified from 75,000 compounds with an IC(50) of 6.5 microM. This method is highly reproducible and reliable for discovery of DPP-IV inhibitors as shown by Z' value of 0.73 and S/N ratio of 6.89.
    Biotechnology Letters 04/2009; 31(7):979-84. DOI:10.1007/s10529-009-9963-y · 1.59 Impact Factor
  • Jin-ling Yang · Hui-xia He · Hui-xin Zhu · Ke-di Cheng · Ping Zhu ·
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    ABSTRACT: The technology of liquid fermentation for producing the recombinant analgesic peptide BmK AngM1 from Buthus martensii Karsch in Pichia pastoris was studied by single-factor and orthogonal test. The results showed that the optimal culture conditions were as follows: 1.2% methanol, 0.6% casamino acids, initial pH 6.0, and three times of basal inoculation volume. Under the above culture conditions, the expression level of recombinant BmK AngM1 in Pichia pastoris was above 500 mg x L(-1), which was more than three times of the control. The study has laid a foundation for the large-scale preparation of BmK AngM1 to meet the needs of theoretical research of BmK AngM1 and development of new medicines.
    Yao xue xue bao = Acta pharmaceutica Sinica 02/2009; 44(1):91-4.
  • Li-Na Wang · Jian-Qiang Kong · Ping Zhu · Guan-Hua Du · Wei Wang · Ke-Di Cheng ·
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    ABSTRACT: X5 protein is one of the putative unknown proteins of SARS-CoV. The recombinant protein has been successfully expressed in E. coli in the form of insoluble inclusion body. The inclusion body was dissolved in high concentration of urea. Affinity Chromatography was preformed to purify the denatured protein, and then the product was refolded in a series of gradient solutions of urea. The purified protein was obtained with the purity of > 95% and the yield of 93.3 mg x L(-1). Polyclonal antibody of this protein was obtained, and Western blotting assay indicated that the X5 protein has the strong property of antigen. Sixty-eight percent of the recombinant protein sequence was confirmed by LC-ESI-MS/MS analysis.
    Yao xue xue bao = Acta pharmaceutica Sinica 12/2008; 43(11):1157-60.
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    ABSTRACT: The expression plasmid pET32CPS harboring SmCPS gene was transformed into E. coli BL21 trxB (DE3) resulting in recombinant strain E. coli [pET32CPS]. The induction of E. coli [pET32CPS] in different temperatures, induction time, IPTG concentrations and A600 values of E. coli were performed. The optimal expression conditions of SmCPS were characterized according to the orthogonal analysis, and the ratio of the interest protein to total proteins reached to 35.6%. The recombinant SmCPS protein purified by Ni2+ affinity chromatography column was identified by SDS-PAGE and Western blotting, and then used for rabbit immunization. The titer of the rabbit antiserum against SmCPS was about 1:24 300 after the third immunization, and could specifically recognize the antigen of SmCPS protein by Western blotting analysis. The successful preparation of polyclonal antibody against SmCPS laid a foundation for further correlative study between expression of SmCPS and the production of tanshinones in protein level.
    Yao xue xue bao = Acta pharmaceutica Sinica 08/2008; 43(7):766-72.