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Expression and Purification of Basic Fibroblast Growth Factor Mutant with Reduced Mitogenic Activity

Xiaoping WU ; Xiaokun LI ; Zhijian SU ; Qing ZHENG ; Sixian WU ; Hua XU ; Hongyan QU

China Biotechnology.2005;25(2):49-52.

In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

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Anticancer Agents from Endophytic Fungi

Min QIU ; Ruisheng XIE ; Yu SHI ; Haimin CHEN

China Biotechnology.2009;29(1):93-104.

Various cancers have seriously threatened human' s health. Screening newer and more effective anticancer agents from natural sources to cure these diseases is the focus in research. As novel sources of potential medicine, a number of metabolites isolated from endophytic fungi have been proved to have anticancer bioactivity. Usually, these endophytic fungi have special biochemical pathway and they can accumulate anticancer agents in cultures such as taxane, alkaloids, cytochalasins, podophyllotoxin, brefeldin A and so forth. The research advance on anticancer agents purified from endophytic fungi is expatiated systemically. In addition, the strategy of screening anticancer agents as well as the prospect on this area is introduced briefly.

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The Global Development of the Biopharmaceutical Industry

Shumei WEN

China Biotechnology.2006;0(01):-.

The number of new medical products based on chemistry launches at the lowest level for many years. The pharmaceutical industry puts emphasis on biotechnology-derived products. Both genomics and protemics are contributing to understanding and determining more targets which involved in human disease.This means that there will be more opportunities for biopharmaceutical breakthroughs,eventually,more and better biomedicines will be approved.Several trends in development for biopharmaceuticals were analyzed:(1) Biological medicine and Chinese traditional medicine have developed rapidly between 1998~2005, the number of the papers about biological medicine increases dramaticly. (2)The emphasis is on biological medicine in pharmaceutical industry and the biotech market is concentrating in United States and multinational. (3)The proportion of biotech products is becoming larger gradually. More concern is focused on biotech products.(4)In order to reduce the cost of R&D and increase the number of new drugs, more and more M&A occur between the biotech companies.(5)The governments all over the world attach importance to biotech products.(6)Tissue engineering, cell therapy and gene therapy have a good future, full of opportunities and challenges.

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Studies on the Effects of Ultrasound on Pepsin,Trypsin and Catalase

Kang WANG

China Biotechnology.2006;0(05):-.

Three kinds of enzymes,pepsin,trypsin and catalase,were selected to research the effects and mechanisms of ultrasound on protein,taking advantage of the change of their biological activity under sonication as the index.The results indicate that the three kinds of enzymes display different modes and degrees of activity change under sonication.In addition,the effects of ultrasound on the enzymes vary with their concentration,which means that the destruction of ultrasound to enzyme could be reduced by adjusting their concentration.Mannitol,a strong free radical scavenger,and Tween-80,a kind of non-ionic surfactant,could protect the activity of the enzymes during sonication.That implies that free radicals,which are produced through the thermal dissociation of water induced by ultrasound cavitation,and the cavitation bubbles,which could disturb the hydrophobic environment around the enzyme molecules,play important roles during the destruction of sonication to enzymes.However,for different enzyme,different factor play the main role.

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Peanut Cotyledon Bioreactor-a Potential New Force in Peanut Industry

Haiyan YAN

China Biotechnology.2006;0(09):-.

The products from bioreactor of peanut cotyledon are organ-specific, enrichment in content, orally taken directly, etc. These properties made it a better bioreactor than others. Current research background and future perspective in this area were introduced. The techniques needed on peanut bioreactor was summarized. The key of peanut bioactor is construction of transformation vector. The promoters of major storage proteins are best choice for construction of transformation vectors. The promoter of glycinin genes from soybean can also be used in construction of transformation vector.

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The Mutasynthetic Production of CHC-B1 from a Streptomyces avermitilis Mutant in the Presence of Cyclohexanecarboxylic Acid

Wei JIANG ; Yang WANG ; Xiaolin ZHANG ; Weiqun GUO ; Di LIU

China Biotechnology.2006;0(08):-.

Gene deletion vector pLJ04(pKC1139∷bkdF+bkdH)was used to disrupt bkdFGH in Streptomyces avermitilis 76-02-e,an industrial producer of anthelmintic avermectin.The disruptants were confirmed by PCR.Shake flask experiment and HPLC analysis showed that the mutant lost the ability to produce avermectins.As it is expected,the mutant,named S.avermitilis bkd76-3,could restore the ability of producing avermectins when the feeding of methylbutyric acid and isobutyric acid to its fermentations was carried out.The addition of cyclohexanecarboxylic acid(CHC)into fermentations of the S.avermitilis bkd76-3 allowed for production of four components,two of which was confirmed as CHC-B1 and CHC-A2 by LC/MS analysis,respectively.

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The Activity Study of Aminodeoxychorismate Synthase of Differernt Corynebacterium glutamicum

Jianhong REN ; Xiaomei ZHANG ; Wenfang DOU ; Hongyu XU ; Zhenghong XU

China Biotechnology.2006;0(08):-.

The two pabAB genes encoding aminodeoxychorismate synthase(ADC synthase)from a L-serine producing strain Corynebacterium glutamicum SYPS-062 and model strain Corynebacterium glutamicum ATCC 13032 were ampilified by PCR.The result of nucleotide sequence analysis showed that both pabAB fragments were 1863bp,encoding 620 amino acids.16 bases differences that resulted in the changes of 7 amino acids were found in the pabAB of SYPS-062.The two pabAB were inserted into pET-28a to yield the recombinant expression vector pET-28a-pabAB and then transfromed into BL21(DE3).Upon IPTG induction,soluble ADC synthase was over-produced by E.coli BL21(DE3)harboring the expression construct.Recombinant ADC synthase purified by Ni-NTA affinity chromatography showed a single band about 67kDa on SDS-PAGE gel,and activity of aminodeoxychorismate synthase analysis show that the enzyme specific activity of SYPS-062 is 46.6% lower than ATCC 13032.

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Tolerance to Low Temperature of Transgenic Aloe Plants with TaDREB Gene from Wheat

Hua ZHAO ; Jin ZHAO ; Yinmao DONG ; Congfen HE ; Qin ZHONG

China Biotechnology.2006;0(09):-.

Plant expression cassette for TaDREB from wheat was constructed into plasmid pBIR1.Aloe stems were used as explants for the transformation mediated by Agrobaterium.Infected tissues were selected using G418 to generate transformants.In total,58 resistant plantlets to the antibiotics were obtained from the infected explants.The designed primers according to the selective gene npt II and the target gene TaDREB were used to analyze all of the G418 resistant plantlets.PCR results demonstrated that TaDREB were successful transferred into aloe genomic with the transformation efficiency of 0.5%.The transgenic aloe plants were treated under 4℃ for two weeks and then at-20℃ for 30min.The treatment showed that the leaves of negative plants appeared severe evidence of freeze injury with brown,withered and translucent,while the positive plants appeared good growing condition.The activities of enzymes such as peroxidase(POD)and superoxide dismutase(SOD)of transgenic plants which were stressed for 14 days under low temperature were analyzed.The results indicated that the trend of SOD and POD activities in transgenic plants was down-up-up-up,and that in non-transgenic plants was down-up-down-down.The average value of relative electrical conductivity in the positive plants was 0.456 which was lower than 0.685 in the negative plants.It is supposed that transformation of the kind of gene could improve the resistant ability of aloe to low temperature.

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Optimization of Fermentation and Induction Conditions of Recombinant E.coli BL21(DE3)/pET30a(+)hrpNEcc

Shu ZHANG ; Min WANG ; Meilin HAN ; Rongcai MA ; Qiang CHEN ; Junlian GAO

China Biotechnology.2006;0(10):-.

In order to obtain high yield of the HrpNEcc protein with a lower total cost,fermentation and lactose induction conditions for recombinant E.coli BL21(DE3)/pET30a(+)hrpN Ecc were optimized in flasks and the recombinant E.coli was fermentated in 7L fermenter.The optimized incoulum concentration was 5% and the optimized nutrient medium was TB medium.The HrpNEcc protein yield reached 417.60mg/L by adding 3g/L exogenous inducer lactose in the growth prophase of log-phase for the recombinant E.coli.The HrpNEcc protein yield was higher 36.73% than that of the case of no any inducer,and was higher 16.85% than that of the case of adding IPTG.The wet weight of cell pellet of the recombinant E.coli reached 57.24g/L after fermentation in 7L fermenter,the HrpNEcc protein reached 3.29g/L,about 50.2% of total cellular protein.

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Analysis of Cellulase Regulator ACEII Interaction with cbh1 Promoter Fragment in Trichoderma koningii

Min LING ; Gang LIANG ; Yongling QIN ; Nan LI ; Zhiqun LIANG

China Biotechnology.2006;0(10):-.

ACEI,ACEII and Xyr1 are transcriptional factors that regulate cellulase gene expression in Trichoderma koningii.In vitro experiments have shown that ACEI and Xyr1 bind to the cbh1 promoter fragment(-304 to-18) and regulate the gene transcription.However,whether ACEII binds to this 287bp fragment is still unclear.To further elucidate the regulatory mechanism of ACEII for cellulase,DNA-binding domains of ACEII from T.Koningii were expressed in E.coli.It could not show binding to the cbh1 promoter fragment(-304 to-18) by electrophoresis mobility shift assays,suggesting that it is Xyr1 but not ACEII binds playing an essential role during induction of cbh1 gene transcription.

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