Improvement of pristinamycin production by genome shuffling and medium optimization for Streptomyces pristinaespiralis

To isolate an improved pristinamycin producing strain of Streptomyces pristinaespiralis, the technique of Genome shuffling was used which resulted in a high-yield recombinant G 3-56 strain. Strain G 3-56 yielded 322 ± 17 mg/L of pristinamycin which was 11.4-fold higher than that of the initial strain and 3.7-fold higher than strain UN-78 which previously had the highest yield of pristinamycin. The genetic characteristics of the recombinant G 3-56 strain was stable as revealed by our subculture experiments. The optimal production medium was determined using the orthogonal matrix method. Under the optimal medium conditions, the maximum yield of pristinamycin was 412 mg/L with about 1.24-fold higher than the original medium.     

野油菜黄单胞菌分泌组双向电泳样品制备方法的建立

野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,Xcc)是十字花科植物黑腐病的病原细菌。我们建立了Xcc的蛋白质组学研究平台,用于分离、鉴定该菌的致病相关蛋白。为了减少胞外多糖(exopolysaccharide,EPS)对蛋白材料质量的影响,我们构建了EPS缺陷的Xcc8004ΔgumB突变体。本研究以Xcc8004ΔgumB出发菌株,用诱导培养液培养,取细胞上清通过超滤浓缩得到蛋白质粗提物,分别采用丙酮沉淀法、试剂盒纯化法和丙酮沉淀-试剂盒联用法来纯化蛋白质粗提物,通过比较双向电泳的结果优选最佳的样品制备方法。结果证明丙酮沉淀-试剂盒联用法较为理想,所得双向电泳图片清晰,分辨率高。因此,此方法可以用于制备野油菜黄单胞菌分泌组双向电泳样品,并可以满足进一步研究的需要。     

BST-2抑制HIV-1病毒样颗粒释放的研究

BST-2是最近发现的可以抑制成熟HIV-1(human immunodeficiency virus,HIV)病毒颗粒从哺乳动物细胞表面释放的宿主因子,随之发现其也可以抑制多种包膜病毒的释放。本研究采用密码子优化的表达HIV-1 gag和gag-pol蛋白的质粒所形成的病毒样颗粒作为研究对象,观测BST-2对这两种病毒样颗粒(Virus-like particle,VLP)的释放抑制情况及其作用机制。结果发现,瞬时表达和稳定表达的BST-2均可以显著抑制病毒样颗粒从哺乳动物细胞释放,同时发现这两种病毒样颗粒(gag/gag-pol)的释放都可以被BST-2抑制;而且,HIV-1中Vpu蛋白可以拮抗BST-2抑制HIV病毒样颗粒释放的作用,另外,通过化学试剂和酶学方法处理,确证BST-2可以被包装进病毒样颗粒中。     

Rapid localization of Gag/GagPol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1

During human immunodeficiency virus type 1 (HIV-1) assembly in HIV-1-transfected COS7 cells, almost all steady-state Gag/Gag and Gag/GagPol complexes are membrane bound. However, exposure to 1% Triton X-100 gives results indicating that while all Gag/GagPol complexes remain associated with the detergent-resistant membrane (DRM), only 30% of Gag/Gag complexes are associated with the DRM. Analysis of the localization of newly synthesized Gag/Gag and Gag/GagPol to the membrane indicates that after a 10-min pulse with radioactive [(35)S]Cys-[(35)S]Met, all newly synthesized Gag/GagPol is found at the DRM. Only 30% of newly synthesized Gag/Gag moves to the membrane, and at 0 min of chase, only 38% of this membrane-bound Gag/Gag is associated with the DRM. During the first 30 min of chase, most membrane-bound Gag/Gag moves to the DRM, while between 30 and 60 min of chase, there is a significant decrease in membrane-bound Gag/Gag and Gag/GagPol. Since the localization of newly synthesized Gag/Gag to the DRM and the interaction of GagPol with Gag both depend upon Gag multimerization, the rapid localization of GagPol to the DRM probably reflects the interaction of all newly synthesized GagPol with the first newly synthesized polymeric Gag to associate with the DRM.     

Co-production of S-adenosyl-l-methionine and glutathione from spent brewer’s yeast cells

Both S-adenosyl-l-methionine (AdoMet) and glutathione (GSH) are important small molecules with pharmaceutical importance. The co-production of AdoMet and GSH using abundant spent brewer’s yeast cells from the beer industry and with l-methionine supplement was successfully realized. Experimental data showed that improvement of GSH productivity was accompanied by AdoMet accumulation. AdoMet productivity of 40–45 mg g⁻¹ (DCW) was successfully achieved and an additional 13–18 mg g⁻¹ (DCW) GSH was synthesized in spent brewer’s yeast cells.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Cloning and expression of the HIV protein in <Emphasis Type="Italic">Escherichia coli</Emphasis> cell-free system

Sixteen kinds of human immunodeficiency virus (HIV) target genes were cloned by polymerase chain reaction (PCR) amplification, and specific plasmids were constructed as the templates for the expression of these genes in the cell-free system. Similarly, the linear PCR templates of these genes for cell-free protein expression were also constructed by using two PCR amplification process. These different templates can be employed to biosynthesize HIV proteins in the cell-free system simultaneously and can be adapted for some high-throughput processes. HIV protease (P10) was performed as a target protein, and two different templates (plasmid and PCR product) were prepared and used for P10 expression in the Escherichia coli cell-free system. The target protein P10 was detected in sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels either by using a plasmid template or by a PCR template. These results are promising and helpful to develop a high throughput process for drug discovery.     

Post-immobilization of modified macromolecular reagents using assembled penicillin acylase for microenvironmental regulation of nanopores and enhancement of enzyme stability

Penicillin acylase (PA) is known to regulate the microenvironment of nanospores. In this study, nanopores containing chemically-modified macromolecules co-assembled with immobilized PA were constructed. We also investigated the various types of functionalized mesocellular siliceous foams (MCFs) commonly used for the immobilization of PA by measuring the catalytic performance and stability of each PA preparation. Amino-MCF activated by p-benzoquinone was chosen as the optimum support for PA immobilization. Successful modification of macromolecules was verified by FT-IR and ultraviolet (UV) spectroscopy. The specific activity of PA co-assembled with dextran 10 k was 99.1 U/mg, which was 1.5-fold that of pristine immobilized PA, while the optimum pH was shifted to neutral. Compared to pristine immobilized and free PA, the optimum temperatures for the modified PA were 5 and 10°C higher, respectively. The residual activity of the ficoll derivative of PA after treatment at 50°C for 6 h was 70%, and this was later increased to 214.5% compared to that of pristine immobilized PA. The dextran 10 k derivative of PA exhibited 90.2% residual activity after 25 times of continuous use. The results show that chemically-modified macromolecules co-assembled with PA in amino-MCF provided a suitable microenvironment for enzyme stability.