Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E. coil

The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed in E. coil in a soluble state. The （His）6 fusion protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography, and the calculated molecular mass（Mr） was 88000. The results of the sequence analysis indicate that the cloned gene（GeneBank accession No. EU124675） was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid. The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides, and further to screen new herbicides.     

Synthesis and Expression in Escherichia coli of a Human Neutrophil Activating Protein-1/Interleukin-8 Gene

The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was sho     

Magnetic nanoparticles used in headspace extraction coupled with DSI-GC-IT/MS for analysis of VOCs in dry Traditional Chinese Medicine

A novel magnetic method using polystyrene modified magnetic nanoparticles to perform thermoheadspace extraction was successfully developed for extraction and preconcentration of volatile organic components in dry Traditional Chinese Medicine(TCM) based on gas chromatography-ion trap/mass spectrometry with a Chromato Probe direct sample introduction device. The dried fruit of Amomum testaceum Ridl. was used as the object TCM. The optimum parameters of headspace magnetic solid-phase extraction were investigated, in which desorption solvent ethyl acetate played a key role in this method,and the headspace extraction temperature of 90℃ and the headspace extraction time of 15 min finally decided. Headspace solid-phase microextraction method was also used to analyze volatile compounds in the TCM to compare with the proposed method. The results show that 60 components were identified totally by two methods; most of the low boiling point chemical compounds are isolated by this new method. In this work, an environmental-friendly and cheap analytical method was established, and a new approach to analyze volatile compounds in dry Traditional Chinese Medicine was also provided.     

Toxicity assessment of heavy metals and organic compounds using CellSense biosensor with E.coli

A new strategy using an arnperometric biosensor with Escherichia coli （E. coli） that provides a rapid toxicity determination of chemical compounds is described. The CellSense biosensor system comprises a biological component immobilized in intimate contact with a transducer which converts the biochemical signal into a quantifiable electrical signal. Toxicity assessment of heavy metals using E.coli biosensors could be finished within 30 min and the 50% effective concentrations （ECso） values of four heavy metals were determined. The results shows that inhibitory effects of four heavy metals to E.coli can be ranked in a decreasing order of Hg^2＋ 〉 Cu^2＋ 〉 Zn^2＋ 〉 Ni^2＋, which accords to the results of conventional bacterial counting method. The toxicity test of organic compounds by using CellSense biosensor was also demonstrated. The CellSense biosensor with E. coli shows a good, reproducible behavior and can be used for reproducible measurements.     

The preparation and fluorescence properties of europium nanoparticles

A new structured metallic nanomaterial of europium nanoparticle was prepared using tannic acid as the reductive agent,and nanoeuropium protein conjugates were synthesized by the method of lipoic acid modification on the surface of nanoparticle,which opens a new field of application of lanthanides in nanotechniques.Their properties were also characterized by UV-vis absorption spectroscopy,transmission electron microscopy (TEM),and fluorescence spectroscopy.The europium nanoparticle and its protein conjugates solution were stable and water-soluble.The fluorescence intensity of the composite europium nanoparticles was significantly increased in the presence of trace protein,and was linear proportional to the concentration of proteins under optimum conditions.According to this,a fluorimetric method for the determination of protein was developed in this paper.     

A Method of Determining Selectivity Coefficients Based on the Practical Slope of Ion Selective Electrodes

It is a problem to be solved that the experimental selectivity coefficients of ion selective electrodes( ISEs) depend on the activity. This paper studied the new method of determining selectivity coefficients. A mixed ion response equation, which was similar to Nicolsky-Eisenman (N-E) equation recommended by IUPAC, was proposed. The equation includes the practical response slope of ISEs to the primary ion and the interfering ion. The selectivity coefficient was defined by the equation instead of the N-E equation. The experimental part of the method is similar to that based on the N-E equation. The values of selectivity coefficients obtained with this method do not depend on the activity whether the electrodes exhibit the Nernst response or non-Nernst response. The feasibility of the new method is illustrated experimentally.     

Chelatometric dispersive liquid-liquid microextraction followed by capillary electrophoresis for the analysis of total and water soluble copper in Rhizoma coptidis

A new method for the analysis of copper and its speciation in Rhizoma coptidis was proposed using chelatometric dispersive liquid-liquid microextraction(cDLLME) followed by capillary electrophoresis. Dithizone was used as a chelating agent for extraction and carbon tetrachloride and ethanol were used as extraction and dispersive solvent,respectively.The calibration curve was linear in the range of 0.5- 20.0μg/mL with a correlation coefficient of 0.9990(n = 6) and the average recovery was in the range of 88.26%-94.44%.The method was successfully used to determine the total and water soluble copper in Rhizoma coptidis herbal medicine.The results suggested that it would be reasonable to setting the criterion of heavy metals in an herbal medicine according to the dosage forms.     

一种新型高比表面积 TiO2载体在 NH3选择性催化还原反应中对 V2O5分散性的促进作用

A titania support with a large surface area was developed, which has a BET surface area of 380.5 m2/g, four times that of a traditional titania support. The support was ultrasonically impregnated with 5 wt%vanadia. A special heat treatment was used in the calcination to maintain the large sur‐face area and high dispersion of vanadium species. This catalyst was compared to a common V2O5‐TiO2 catalyst with the same vanadia loading prepared by a traditional method. The new cata‐lyst has a surface area of 117.7 m2/g, which was 38%higher than the traditional V2O5‐TiO2 catalyst. The selective catalytic reduction (SCR) performance demonstrated that the new catalyst had a wid‐er temperature window and better N2 selectivity compared to the traditional one. The NO conver‐sion was>80%from 200 to 450 °C. The temperature window was 100 °C wider than the traditional catalyst. Raman spectra indicated that the vanadium species formed more V‐O‐V linkages on the catalyst prepared by the traditional method. The amount of V‐O‐Ti and V=O was larger for the new catalyst. Temperature programmed desorption of NH3, temperature programmed reduction by H2 and X‐ray photoelectron spectroscopy results showed that its redox ability and total acidity were enhanced. The results are helpful for developing a more efficient SCR catalyst for the removal of NOx in flue gases.     

Iodide analysis by ion chromatography on a new stationary phase of polystyrene-divinylbenzene agglomerated with polymerized-epichlorohydrin-dimethylamine

A new stationary phase for iodide ion analysis has been developed. The cationic polymerepichlorohydrin-dimethylamine(PEPI-DMA) was served as modifier in synthesizing polyelectrolyte sorbents and the macroporous polystyrene-divinylbenzene(PS-DVB) resin was used as support. The positively charged polymer(PEPI-DMA) was electrostatically bonded to a negatively charged particle(PS-DVB sulfonated). The new stationary phase was characterized by scanning electron microscopy(SEM), Fourier transform infrared(FTIR), elemental analysis, chemical adsorption and desorption measurements. The chromatographic evaluation of the new stationary phase was performed using various anions with a conductivity detector. The new stationary phase was also applied to the determination of iodide directly with a DC amperometric detector using a platinum working electrode and an Ag/Ag Cl reference electrode. The chromatographic conditions were optimized and the eluent solution contained 5 mmol/L HNO3 and 15 mmol/L Na NO3 at a flow rate of 1.0 m L/min and column temperature of 30 8C. The applied voltage of the DC amperometric detector was 0.9 V. Under the optimum conditions, the linear range of the method was 0.2–50 mg/L for iodide ion with a correlation coefficient of 0.9990. The detection limit was 0.05 mg/L(calculated at S/N = 3) and the relative standard deviations(RSD, n = 6) were all less than 1% for retention time, peak area and peak height. This method was also utilized for the determination of iodide ions in samples of povidone iodine solution and kelp samples with satisfactory results.     

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-△SHP-1 Antibodies

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated △SHP-1) and the preparation of its polyelonal antibodies.A cDNA fragment encoding △SHP-1 was amplified by PCR and then cloned into the pT7 expression vector.The recombinant pT7-△SHP-1 plasmid was used to transform Rosetta(DE3) E.coll cells.△SHP-1 was distributed in the exclusion body of E.coll cell extracts and was purified through a two-column chromatographic procedure.The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns.It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases.To generate polyclonal anti-△SHP-1 antibodies,purified recombinant △SHP-1 was used to immunize a rabbit.The resultant anti-serum was subjected to purification on △SHP-1 antigen affinity chromatography.The purified polyclonal antibody displayed a high sensitivity and specificity toward △SHP-1.This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.     

Basic studies of hydrogen evolution by escherichia coli containing a cloned citrobacter freundii hydrogenase gene

Citrobacter freundii genes that complemented Escherichia coli hyd-(hydrogenase activity) mutation were cloned in plasmids pCBH4 (6.2 kb) and pCBH6(5.7 kb). Hydrogen evolution by the transformant E. coli HK-8(pCBH4 or pCBH6) was investigated. The optimum culture temperature of recombinant E. coli cells for hydrogen evolution from glucose was in the neighborhood of 18 degrees C. The recombinant E. coli cells cultured at this condition showed a several-fold increase of hydrogen evolution, as compared with that of the wild-type cells. The plasmid-retention stability of this recombinant E. coli was extremely high, especially plasmid pCBH4, which was completely retained during 2 wk without any restriction. Hydrogen production by immobilized recombinant E. coli was then investigated using cells cultured at 18 degrees C. The hydrogen evolution rate from glucose and Lennox-broth were about twofold higher than that of E. coli C600, and this high hydrogen evolution rate was maintained for more than 1 mo.     

利用微生物直接生产羟基丁酸单体

利用重组大肠杆菌E.coli HB101来进行直接生产羟基丁酸(HB)手性单体,研究了含pUCAB质粒的重组体E.coli HB101在各种条件下生长及积累HB单体的情况,研究了HB单体的积累随pH值变化的规律。结果表明,pH=6.8时,48 h内细菌可生产0.5 g/L以上的HB单体。     

产甘油基因工程菌的构建

利用途径工程的基本原理,在大肠杆菌中构建一条产甘油的新代谢途径。从酿酒酵母Sacchdromyces cerevisiae INVSc1菌株总DNA克隆3-磷酸甘油脱氢酶基因(gpdl)和3-磷酸甘油酯酶基因(hot2),构建由两个杂合启动子trc启动基因的双表达盒的重组质粒pGEM-Cgpd1-Chor2,后者转入E．coli JM109菌株,构建的重组菌株就具有一条直接将葡萄糖转化为甘油的新代谢途径,将该重组菌株以葡萄糖为底物进行摇瓶发酵,甘油产率为1．18g／L。该研究结果为进一步构建生产1,3-丙二醇工程菌打下了基础。     

Ligation independent cloning (LIC) as a rapid route to families of recombinant biocatalysts from sequenced prokaryotic genomes

A technique is presented for the high throughput generation of families of recombinant biocatalysts sourced from prokaryotic genomes, providing rapid access to the naturally evolved diversity of enzyme specificity for biocatalyst discovery. The method exploits a novel ligation independent cloning strategy, based on the locally engineered vector pET-YSBLIC and has been used for the rapid generation of a suite of expression plasmids containing genes encoding a family of six Baeyer-Villiger monooxygenases (BVMOs) from Mycobacterium tuberculosis H37Rv (MTb). The six resultant recombinant strains of E. coli B834 (DE3) expressing the genes were assayed for oxygenating activity in respect of the target reaction; the resolution of bicyclo[3.2.0]hept-2-en-6-one. The analysis of biotransformations catalysed by growing cells of E. coli was complicated by the production of indole in the reaction mixtures, possibly resulting from the in vivo activity of E. coli tryptophanase. Four of the recombinant strains expressing different BVMOs catalysed the oxidation of one or more of four screening substrates, well above controls that had been transformed with the re-ligated parent vector. One of the recombinant strains, E. coli B834 (DE3) pDB5, expressing the Rv3049c gene from MTb, was found to effectively resolve the target substrate, yielding a 19% yield of (1R, 5S)-(+)-bicyclo[3.2.0]hept-2-en-6-one with >95% enantiomeric excess in a 4 L fermentation reaction.     

来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni²⁺柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.     

猪IL-18成熟蛋白在大肠杆菌中的表达及生物学活性鉴定

以含猪IL-18全基因的重组质粒pGEM-IL-18为模板,PCR扩增猪IL-18成熟蛋白基因.将IL-18成熟蛋白片段定向插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-IL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白(His-IL-18),并进行融合蛋白的纯化、生物学活性鉴定.结果表明,SDS-PAGE可检测到相对分子质量约为2.1×104的融合蛋白,westem blot证实His-IL-18能与猪IL-18单克隆抗体发生特异性反应.重组猪IL-18经纯化后,能明显刺激猪脾脏T淋巴细胞增殖反应,在Marc-145细胞上抗猪繁殖与呼吸综合征病毒的活性为2.50×103IU/mg,在PK-15细胞上抗猪伪狂犬病毒、猪细小病毒的活性分别为2.00×103和2.24×103IU/mg.表明建立的表达系统能够表达重组猪IL-18,表达的重组猪IL-18具有一定的生物学活性.     

THE EXPRESSION OF ENTEROTOXIN A~-B~+ GENE OF V. cholerae IN E. coli

The cloning in E. coli of a cholerae toxin gene that is A~-B~+ has been successfully constructed by using DNA recombinant techniques. E. coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins.     

The expression of enterotoxin A-B+ gene of V. cholerae in E. coli

The cloning in E. coli of a cholerae toxin gene that is A-B+ has been successfully constructed by using DNA recombinant techniques. E. coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins.