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Purification of a New Antibacterial Protein from Housefly(Musca domestica)Larvae

Hui QU ; Youjin HAO ; Yingjun JING ; Desen LI ; Rongqian DU

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

Insects exhibit a particular resistance to infections.The activation of the innate immune response of insects is involved in the recognition of the infectious nonself and subsequent activation of cellular and humoral reactions.In humoral reactions,insect antibacterial peptides and lysozyme are very important in resistance to infections.Housefly(Musca domestica) is one of the most important kinds of insects and it has strong ability to adapt to the adverse circumstances.It is of momentous theoretical and practical significance to research the immunity system of housefly.The methods of inducement of housefly larvae were firstly studied.Then an antibacterial protein,whose molecular weight is 28kDa,was purified from housefly larvae,induced by 30% H2O2,through saltout,Sephadex G25 column,Sephadex G75 column and CMSepharose Fast Flow column.This antibacterial peptide had activities against most of Gram positive and Gram negative bacteria.

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Construction and Determination of an Episomal Secreting Expression Vector:pYES2/CT/?-factor for Saccharomyces cerevisiae

Yadong HU ; Dongsheng YAO

China Biotechnology.2006;0(12):-.

pPIC9 was used as a template and ?-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/?-factor(pYC?)was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man(pKLman) was cut by restriction enzymes and linked with pYC?.This recombinant vector pYC?-man was used to determine the secretory ability and stability of pYC?.The excellent secretory ability of pYC? was proved by two experiments.One showed that INVSc1/pYC?-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYC? clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYC? were not detected,while INVSc1/pYC?-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYC? was also very good proved by continuous cultivation for about 150h.

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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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Agrobacterium-mediated Transformation of an Economically Important Potato Cultivar Using Internodal Stem Explants

Li TANG ; Hui TANG ; Suying WANG ; Xiaoli YANG ; Haengsoon LEE ; Sangsoo KWAK

China Biotechnology.2007;27(7):80-87.

Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, short duration (7～8 weeks), high efficiency and easy process make this system well suited for wider commercial applications of transgenic Atlantic potato plants.

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Computational Identification of microRNAs and Their Targets

Zhiyun GUO ; Canquan MAO ; Lili XIONG

China Biotechnology.2008;28(10):118-123.

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Since inception, computational methods have been an invaluable tool complementing experimental approaches, and many discoveries have been obtained through combination of experimental and computational approaches. The knowledge that has been accumulated about the principles of miRNAs and target recognition were reviewed. The currently available computational methodologies and software for prediction of miRNA and their target genes also have been discussed.

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Detection and Partial Characterization of γ-glutamyltranspeptidase from Cordyceps sinensis Mycelia

Jianglan YUAN ; Xu KANG ; Zheng HU ; Guolin ZOU

China Biotechnology.2008;28(10):100-105.

γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

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Purification and Characterization of One Alkaline Protease from Actinomucor elegans AS3.2778

Jinquan PAN ; Xiaochun LUO ; Mingquan XIE

China Biotechnology.2008;28(9):111-118.

One alkaline protease from Actinomucor elegans AS3.2778 was purified protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.

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Molecular Cloning and Expression of Cardiotoxin Ⅲ from Naja naja atra in E.coli and Yeast Pichia pastoris

Xingyong CHEN ; Ping LU ; Jing LIU ; Kangsen XU

China Biotechnology.2008;28(8):23-30.

Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.

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Expression, Purification and Characterization of [Gly14]-Humanin, a Novel Neuroprotective Peptide

Baofeng YU ; Jun XIE ; Xianjiu CHEN ; Yuehong ZHANG ; Huizhen WANG ; Niuliang CHENG ; Bo NIU

China Biotechnology.2008;28(4):21-26.

Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.

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