Tandem Repeat Expression and Inhibitory Activity of a ?-lactamase Inhibitory Peptide / 中国生物工程杂志

?-lactamase inhibitor research is popular for its potential on ?-lactam antibiotics resistant strain.A ?-lactamase binding peptide SIPIS04-01 was obtained by the yeast two-hybid system.In vitro assay showed that it can inhibit the ?-lactamase activity.In order to improve the expression level of the recombinant peptide,a two-copy expression plasmid pYG563 was constructed by random orientation tandem repeat method after codon modification,the two-copy plasmid was successfully expressed and the product was increased by 48.4% than that of one-copy plasmid.Purified peptide showed inhibitory activity against TEM-1 ?-lactamase in vitro and the inhhibitory constant Ki was measured.

Expression and Characterization of Thrombin Inhibitor TTI / 中国生物工程杂志

TTI gene coding for Tsetse thrombin inhibitor was modified with E.coli bias codon and expressed in Escherichia coli with high efficiency.Recombinant protein was purified to more than 98% purity.Assay for enzyme activity determination was set up.The result showed that the fusion protein exhibited inhibiting activity for thrombin.Inhibitory rate of purified TTI was 73% when concentration of thrombin and substrate was 10U/ml and 250?mol/L respectively.Inhibition pattern was determined as competitive with Ki at 35?mol/L.

Cloning and Expression of dauW Gene from Daunorubicin-producing Streptomyces coeruleorubidus SIPI-1482 / 中国生物工程杂志

A novel gene,located between dnrX and drrB in the genome of daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482,was cloned and named as dauW.The full sequence of dauW was submitted to GenBank(Accession No.EF523565).Blast result indicated that it showed high homology with dnrW in GenBank.The exact function of dauW is as yet unknown despite the possibility that it might belong to a family of FAD-dependent oxidoreductases on the basis of conserved domain analysis.dauW was cloned into expression plasmids pET-28a(+)and pET-32a(+),respectively,and was successfully expressed in E.coli DE3 after induction with IPTG.The preliminary results of the expression of dauW suggested that it might be involved in the self resistance in Streptomyces coeruleorubidus due to the increased resistance to daunorubicin in the E.coli host.

Application Progress of Vitreoscilla Hemoglobin / 中国生物工程杂志

Vitreoscilla Hemoglobin(VHb), with the function of increasing the growth of and product yield by a heterologous host, has been widely use in the area of fermentation, environment protection, transgenic animal and plant, recombinant protein expression, etc. Fusion protein of VHb with other enzyme or protein can enhance activity and stability of the enzyme or isolation efficiency of the protein. The reconstitution of VHb will be helpful to obtain ‘novel’ proteins which have better activity.

DNA Shuffling of Vitreoscilla Hemoglobin / 中国生物工程杂志

To improve the growth enhancement activity of Vitreoscilla hemoglobin(VHb), Vitreoscilla hemoglobin gene(vgb) was mutated by error-prone PCR and then reconstituted by DNA shuffling. The shuffling library was constructed by inserting the shuffled genes into the downstream of vgb natural promoter and transforming them into E.coli DH5?. Mutated active VHb proteins were first screened in test tubes according to host cell pellets color and then in shake flasks according to host pellets wet weight .One active mutant protein, VHb′042506, was obtained after second screening. It could increased the host wet weight by 31.25% and 58.75% than that of the control which bearing natural VHb under microaerobic and extremely microaerobic conditions, respectively. Sequencing and alignment results showed that 11 nucleotides were mutated, thus resulted in 4 amino acids changes occurred in this mutant protein. CO difference spectrum test also indicated that it had higher specific absorption.

Disruption and Compensation of dnmV Gene from Daunorubicin-producing Strain Streptomyces coeruleorubidus SIPI-1482 / 中国生物工程杂志

TDP-4-ketohexulose reductase, encoded by dnmV, is important in daunorubicin biosynthesis. To obtain a daunorubicin block mutant, double cross-over plasmid pYG817 was constructed by inserting apramycin resistant gene and amplified dnmV together with upstream dnmU into vector pUC18. dnmV was successfully disrupted after transformation of daunorubicin-producing strain SIPI-1482 by pYG817. Daunorubicin was absent from metabolites of the resulting transformant, and its biosynthesis can be reconstituted by introducing dnmV expression plasmid into the disruptant, although the yield is lower than wild-type SIPI-1482, according to HPLC analysis. This mutant can be a good candidate for production of anthracycline such as epi-daunorubicin by introducing exogenous gene into the host.

A Convenient Method to Select DNA Fragments of Cephalosporium acremonium with Promoter Function / 微生物学通报

A promoter-trap vector pGBT14 for selecting promoters of fungus gene was constructed with E. coli-yeast shuttling plasmid pGBT9. Using this vector, a0. 5-2. 0kb chromosomal DNA library of Cepholosporium acremonium was constructed, and twenty four DNA fragments with promoter function in Saccharomyces oerevisiae Y153 were selected from this DNA library. And the promoter function of these DNA fragments was analyzed.