Ligands of TetR family transcriptional regulators: a review / 生物工程学报

TetR family transcriptional regulators (TFRs) are widely distributed in bacteria and archaea, and the first discovered TFR was confirmed to control the expression of tetracycline efflux pump in Escherichia coli. TFRs can bind DNAs and ligands. Small molecule ligands can induce conformational changes of TFRs, inhibiting or promoting TFRs to control target gene expression. Currently, TFRs have a wide variety of ligands, including carbohydrates, proteins, fatty acids and their derivatives, metal ions, and so on. Due to the diversity of ligands, TFRs regulate a wide range of physiological processes, from basic carbon metabolism and nitrogen metabolism to quorum sensing and antibiotic biosynthesis. On the basis of the recent studies in our laboratory and the literature, we review here the regulatory mechanism mediated by ligands of TFRs in primary and secondary metabolism, as well as the application of ligands for TFRs in the development of gene route and the activation of antibiotic biosynthesis.

Screening of host proteins interacting with GP, VP30, NP,and L proteins of Ebola virus by yeast two-hybrid system / 军事医学

Objective To study the biological function of GP,VP30,NP and L proteins in Ebola virus(EBOV) infectious diseases,and to screen the host proteins which interact with GP,VP30,NP,and L proteins of EBOV from human liver cDNA library using yeast two-hybrid technique.Methods Recombinant PCR was used to construct bait plasmids pGBKT7-GP,pGBKT7-VP30,pGBKT7-NP and pGBKT7-L.Bait strains were combined with human liver cDNA library strains.The positive clones were analyzed by DNA sequencing and bioinformatics.A yeast recovery experiment was performed to further verify and exclude false positive results.Results We constructed bait plasmids pGBKT7-GP,pGBKT7-VP30,pGBKT7-NP and pGBKT7-L with the recombinant PCR method.Six host proteins which could interact with GP,VP30,NP,and L proteins were screened,including COMMD1,ALB,PSMD8,APOA2,CYP2E1,and HP.The yeast recovery experiment proved that COMMD1 and APOA2 might interact with NP protein.Conclusion A number of prey proteins which interact with GP,VP30,NP,and L proteins of EBOV are screened,which may provide reference for the research of EBOV.

Separation of human γ-tubulin ring complexes from cell lysates using gel filtration chromatography / 军事医学

Objective To separate human γ-tubulin ring complexes (γTuRC) .Methods Cell lysates prepared from 293FT cells were separated using gel filtration chromatography .Then, the eluate fractions containing γTuRC or γ-tubulin small complexes (γTuSC ) were determined by immunoblotting .Results As the constitutive components of γTuRC,γ-tubulin,γ-tubulin complex protein 2 (GCP2), GCP3 and GCP4 were eluted and enriched in the fourth fraction .The molecular mass of eluates in the fourth fraction was about 2000 ×10 3 .Following γTuRC, the constitutive components ofγTuSC including γ-tubulin, GCP2 and GCP3 were eluted and enriched in the fourteenth fraction .The molecular mass of eluates in the fourteenth fraction was about 310 ×10 3 .Unassembled free components were washed out in the eighteenth and subsequent fractions .γTuRC could be detected in the corresponding fractions by negative-PAGE separation .ConclusionγTuRC and γTuSC were successfully separated from the unassembled free components in the fourth ( 4#) and fourteenth (14#) eluted fraction, respectively.The eluates containing ofγTuRC orγTuSC can be used for microtubule assembly research.

Optimized sample preparation for metabolome studies on Streptomyces coelicolor / 生物工程学报

Streptomycetes produce many antibiotics and are important model microorgansims for scientific research and antibiotic production. Metabolomics is an emerging technological platform to analyze low molecular weight metabolites in a given organism qualitatively and quantitatively. Compared to other Omics platform, metabolomics has greater advantage in monitoring metabolic flux distribution and thus identifying key metabolites related to target metabolic pathway. The present work aims at establishing a rapid, accurate sample preparation protocol for metabolomics analysis in streptomycetes. In the present work, several sample preparation steps, including cell quenching time, cell separation method, conditions for metabolite extraction and metabolite derivatization were optimized. Then, the metabolic profiles of Streptomyces coelicolor during different growth stages were analyzed by GC-MS. The optimal sample preparation conditions were as follows: time of low-temperature quenching 4 min, cell separation by fast filtration, time of freeze-thaw 45 s/3 min and the conditions of metabolite derivatization at 40 degrees C for 90 min. By using this optimized protocol, 103 metabolites were finally identified from a sample of S. coelicolor, which distribute in central metabolic pathways (glycolysis, pentose phosphate pathway and citrate cycle), amino acid, fatty acid, nucleotide metabolic pathways, etc. By comparing the temporal profiles of these metabolites, the amino acid and fatty acid metabolic pathways were found to stay at a high level during stationary phase, therefore, these pathways may play an important role during the transition between the primary and secondary metabolism. An optimized protocol of sample preparation was established and applied for metabolomics analysis of S. coelicolor, 103 metabolites were identified. The temporal profiles of metabolites reveal amino acid and fatty acid metabolic pathways may play an important role in the transition from primary to secondary metabolism in S. coelicolor.

Enhanced erythromycin production in Saccharopolyspora erythraea by tandem expres-sion of metK, vhbS and adpA / 军事医学

Objective To construct erythromycin-overproducing mutants by tandemly expressing S-adenosylmethionine synthetase gene metK, Vitreoscilla hemoglobin gene vhbS and pleiotropic regulatory gene adpA in Saccharopolyspora eryth-raea.Methods Through PEG-mediated protoplast transformation , the integrative plasmid carrying metK, vhbS and adpA was respectively introduced into erythromycin-producing wild-type strain S.erythraea A226 and industrial strain WB .The engineered strains were generated by apramycin resistance screening and PCR identification .The erythromycin production was compared in original strains and their mutants by the inhibition test of Bacillus subtilis and HPLC analysis .Results and Conclusion Four A226-derived mutants A226-P1-P4 and three WB-derived mutants WB-P1-P3 were independently obtained.Compared with wild-type strain A226, the relative erythromycin titer of the four engineered strains A 226-P1-P4 was increased from 8%to 25%by scoring the growth-inhibition zones .Further HPLC analysis showed that the four mutants had increased erythromycin A yield by 64%-94%.Likewise, the relative erythromycin titer and erythromycin A yield of the three engineered strains WB-P1-P3 were enhanced by 6%-10%and 31%-62%, respectively, in comparison with the original strain WB.The results show the universality of enhancing erythromycin productionvia tandem expression of metK, vhbS and adpA in S.erythraea.

Design and activity analysis of chimeric epidermal growth factor fusion vaccine E5T-mSEA / 生物工程学报

Epidermal growth factor receptor (EGFR) and its ligands (EGF and TGFalpha) are over-expressed in a variety of tumors. Immunization EGF-carrier protein inhibits tumor growth through abrogating binding of EGF to EGFR. Here, a chimeric protein of EGF and TGFalpha (E5T) was genetically fused to Staphylococcal enterotoxin A (SEA), a bacterial superantigenic protein which promotes humoral B cell response through enhancement of Ag-specific CD4 T cells activity. The resulted fusion proteins were expressed in Escherichia coli and purified though metal chelating affinity chromatography. Immunization of E5T-mSEA fusion protein in mice induced production of high titers antibodies, which recognize both EGF and TGFalpha. Anti- E5T-mSEA serum at dilution of 1:10 significantly inhibited growth of A431 cell lines but had little effect on 293T cell lines.