Cloning and Expression of Rat Liver S-Adenosylmethionine Synthetase

Introduction S Adenosylmethionine(SAM)isabiologicallyac tivecompoundwidelydistributedinthebodytissues andfluids.Itisinvolvedinanumberofbiochemical reactions.Asamethylgroupdonor,itisimportantin transmethylationreactions,whichcontributestothe synthesesofsuc…     

Purification, cloning and expression of an Aspergillus niger lipase for degradation of poly(lactic acid) and poly(ε-caprolactone)

A lipase from Aspergillus niger MTCC 2594 was purified 53.8-fold to homogeneity by hydrophobic interaction chromatography using octyl sepharose and the enzyme showed two protein bands with apparent molecular mass of 35 and 37 kDa respectively. The lipase exhibited maximum activity at pH 7.0 and 37 °C and was stable between pH 4.0 and 10.0 and temperatures up to 50 °C. The values of K_m and V_max were 3.83 mM and 32.21 μmol/min/mg respectively, using olive oil as substrate. Lipase encoding gene, lipA, coded for 297 amino acid residues with conserved pentapeptide sequence, G-H-S-L-G, was cloned and expressed in Pichia pastoris. Although lipA showed high homology with the known Aspergillus lipases, it exhibited differences in putative lid domain. Both native and recombinant lipases have potential for degradation of poly(lactic acid) and poly(ε-caprolactone), and the present study will serve as a baseline of initial studies for its exploitation in polymer degradation.     

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.     

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.     

Construction of a yeast plasmid cloning vector with high stability inSaccharomyces cerevisiae strains deficient in 2 µmDNA

PlasmidpCP1 was constructed from cloned 2 µmDNA ofSaccharomyces cerevisiae and from plasmidpJDB207. VectorpCP1 contains the completeB form of 2 µmDNA interrupted in theFLP gene, together withDNA derived from theEscherichia coli plasmidpAT153 and a low expression variant of theS. cerevisiae LEU2 gene. The new vector is lost at a low frequency from yeastcir ⁺ orcir ⁰ strains under non-selective growth conditions and is stable against rearrangements incir ⁰ strains. Its usefulness for curingcir ⁺ strains from endogenous 2 µmDNA and for their conversion tocir ⁰ strains was demonstrated.Dedicated to Professor Dr.Karl Schlögl on the occasion of his 60th birthday.     

cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

Introduction Theleukemiainhibitoryfactor(LIF)isamulti functionalcytokinebelongingtotheinterleukin6(IL6)family[1].Itwasfirstfoundasamyeloidleukemic cellproliferationsuppressoranddifferentiationinducer cytokine[2].ThenaturalformofLIFisa38to67kDa glycosylate…     

Recombination System Based on Cre α Complementation and Leucine Zipper Fusions

A gene encoding β-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated molecular weight of 24.44 kDa. The β-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel–nitrilotriacetic acid (Ni–NTA) column. The purified β-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml^-1 activities for the substrates of barley β-glucan and lichenan, respectively, and the specific activities were 728.79 and 379.1 U mg^-1 for them, respectively. The optimal temperature and pH of the purified enzyme were 40°C and 6.4, respectively. When barley β-glucan was used as the substrate, K _m was 5.34 mg ml^-1, and K _cat showed 7,206.71 S^-1, thus the ratio of K _cat and K _m was 1,349.67 ml s^-1 mg^-1. The activity of β-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA).     

Molecular Cloning of a Glycyrrhiza uralensis F. Aquaporin GuPIP1 Up-regulated in Response to Drought, Salt and ABA Stress

IntroductionWater is an ubiquitous and indispensable moleculefor plant growth and development[1]. According to thecomposite water flux model[2], water is transported intoplant tissues by three pathways: apoplastic, symplas-tic, and transcellular. The latt…     

Information cloning of harmonic oscillator coherent states

We show that in the case of unknown harmonic oscillator coherent states it is possible to achieve what we call perfect information cloning. By this we mean that it is still possible to make arbitrary number of copies of a state which has exactly the same information content as the original unknown coherent state. By making use of this perfect information cloning it would be possible to estimate the original state through measurements and make arbitrary number of copies of the estimator. We define the notion of a measurement fidelity and calculate it for our case as well as for the Gaussian cloners.     

ltB-ureB融合基因原核表达系统的构建及其产物免疫性和佐剂活性的鉴定

构建ltB-ureB融合基因原核表达系统并对其表达产物的免疫性和佐剂活性进行鉴定.采用PCR和T-A克隆法从幽门螺杆菌(Helicobacter pylori,Hp)临床菌株Y06和大肠杆菌44851株DNA中获得了ureB和ltB全长基因扩增片段及其克隆,并构建了ltB-ureB融合基因及其原核表达系统pET32a-ltB-ureB-E.coli BL21DE3.在E.coli BL21DE3宿主菌中用不同浓度的IPTG诱导表达,并用Hp全菌抗体的Western blot、ELISA以及GM1ELISA分别证实了目的重组蛋白(rLTB-UreB)的免疫性和佐剂活性.与报道的相关序列比较,所克隆的ureB和ltB核苷酸序列同源性分别为96.88%～97.82%和99.12%～99.71%,氨基酸序列同源性为99.65%～99.82%和97.58%～99.19%.0.1～1.0 mmol/L的IPTG均能有效地诱导目的重组蛋白rLTB-UreB的表达,该蛋白主要以包涵体形式存在,其产量约为细菌总蛋白的35%.Western blot结果证实rLTB-UreB不仅能与商品化的Hp全菌抗体结合,免疫家兔后也能产生特异性抗体,表明rLTB-UreB有良好的免疫反应性及抗原性.GM1-ELISA结果显示rLTB-UreB能与牛GM1结合,表明rLTB-UreB有佐剂活性.以兔抗rLTB-UreB为一抗,发现所检测的109株Hp临床分离菌株均表达UreB;以rLTB-UreB为包被抗原,发现所检测的125例Hp感染者血清中均存在UreB抗体;表明UreB广泛存在于不同的Hp菌株中,并有很强的抗原性,也提示rLTB-UreB确有自然表达UreB的抗原特异性.本文成功地构建了LTB-UreB融合基因原核高效表达系统,所表达的LTB-UreB融合蛋白有良好的免疫性和佐剂活性,为Hp基因工程疫苗的产业化奠定了坚实的基础.