Development of broad-host-range vectors for expression of cloned genes in Pseudomonas

The cloning and expression of genes in Pseudomonas have been difficult, until now, due to the absence of vector systems that contain multiple restriction sites downstream from promoter sequences that are functional in Pseudomonas. We report here the construction of several broad-host-range vectors that can be utilized in either Pseudomonas or Escherichia coli and that rely on easily selectable antibiotic resistance markers with multiple cloning sites. These vectors were constructed by inserting the entire pUC13 sequence into derivatives of the RSF1010 wide-host-range plasmid. From this construction, other derivatives were obtained, specifically a lacZ::KmR fusion gene which provides an easily selectable marker in both E. coli and Pseudomonas. These vectors have been used to express the Pseudomonas putida cytochrome P450 monoxygenase gene in a P450-deficient P. putida strain. Thus, these vectors allow for the cloning, expression and selection of Pseudomonas genes in Pseudomonas by complementation.     

串联亲和纯化原核表达载体的构建

【目的】构建串联亲和纯化原核表达载体,用于研究细菌中(生理状态或接近生理条件下的)蛋白-蛋白相互作用。【方法】设计并合成两条串联亲和标签序列,分别可以在靶蛋白N端和C端融合Protein G和链亲和素结合肽(Streptavidin binding peptide,SBP)标签;以pUC18载体为骨架,去除原有的阻遏蛋白基因,构建组成型表达载体pNTAP和pCTAP。【结果】成功构建N端和C端标签表达载体pNTAP和pCTAP,它们在大肠杆菌(Escherichia coli)BL21(DE3)、肠出血性大肠杆菌O157:H7和痢疾杆菌福氏5型M90T菌株中都可以实现表达。【结论】本实验构建的两个串联亲和纯化表达载体可以在部分革兰氏阴性细菌中表达,为研究细菌内蛋白-蛋白相互作用及致病菌毒力蛋白的作用机制奠定了基础。     

Construction of broad-host-range vectors for the selection of divergent promoters

A series of promoter-probe plasmid vectors has been constructed which allows for the selection of DNA sequences containing divergent control elements. Each vector contains a pair of promoterless genes [encoding beta-galactosidase (lacZ), alkaline phosphatase (phoA), and bacterial luciferase (luxAB)] arranged in an antiparallel fashion and separated by a large intervening multiple cloning site. The vectors permit direct detection of promoter activity on indicator plates after transformation. Cloned promoters are selected based on production of coloured products in the case of lacZ and phoA, and by the emission of light in the case of luxAB. These vectors have been tested using known divergent promoter elements from pBR322 and Pseudomonas phage D3.     

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.     

Cloning technologies for protein expression and purification

Detailed knowledge of the biochemistry and structure of individual proteins is fundamental to biomedical research. To further our understanding, however, proteins need to be purified in sufficient quantities, usually from recombinant sources. Although the sequences of genomes are now produced in automated factories purified proteins are not, because their behavior is much more variable. The construction of plasmids and viruses to overexpress proteins for their purification is often tedious. Alternatives to traditional methods that are faster, easier and more flexible are needed and are becoming available.     

Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters.

We have constructed a series of broad-host-range plasmids which use "visual screens" to detect promoter activity. These plasmids contain the pMB1 and pRO1600 origins of replication and are capable of replicating in a wide range of gram-negative bacteria. The genes encoding beta-galactosidase and alkaline phosphatase from Escherichia coli and bacterial luciferase from Vibrio harveyi supply the promoterless indicator genes. The constructs were tested in E. coli and Pseudomonas aeruginosa.     

Construction of broad-host-range vectors for general cloning and promoter selection in Pseudomonas and Escherichia coli

We have constructed two promoter-selection vectors based upon the broad-host-range plasmid pRO1614. pQF40 (6 kb) contains a promoterless tetA gene downstream from a large multiple cloning site while pQF26 (5.4 kb) possesses a promoterless cat cartridge. The latter vector displayed a copy number of 13 in Pseudomonas aeruginosa and 39 in Escherichia coli. When promoter sequences derived from the Pseudomonas phage phi PLS27 were cloned into pQF26, high levels of chloramphenicol-acetyltransferase were detected in P. aeruginosa. In E. coli the activity was approximately one-third that in P. aeruginosa when corrections were made for the plasmid copy number.     

A generic protein purification method for protein complex characterization and proteome exploration.

We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.     

人同型融合和蛋白质分选复合体亚基的原核表达、蛋白纯化及功能验证

同型融合和蛋白质分选复合体(HOPS)由VPS11、VPS16、VPS18、VPS33、VPS39和VPS41这6种蛋白组成,能够通过膜融合机制来调节生物体内的膜泡运输。已有研究表明其可以作为融合因子来促进自噬体与溶酶体膜融合过程。为在体外确定HOPS复合体与自噬性SNARE蛋白STX17是否具有直接相互作用,首先利用PCR技术从已有质粒中扩增得到6种基因的编码序列,将其连接至pGEX 4T-1-GST或pET-His-NusA原核表达载体上,经菌落PCR初步鉴定和DNA测序无误后成功构建6种原核表达重组质粒并转化至大肠杆菌BL21(DE3);利用谷胱甘肽琼脂糖树脂与镍柱对重组蛋白进行纯化,烟草蚀纹病毒(TEV)蛋白酶酶切掉GST或His-NusA标签,得到分子量约为105 kDa的HA-VPS11蛋白、97 kDa的Flag-VPS16蛋白、108 kDa的HA-VPS18蛋白、70 kDa的Flag-VPS33蛋白、97 k Da的HA-VPS39蛋白和98 kDa的Flag-VPS41蛋白;通过体外GST pull-down技术对6种蛋白的功能进行验证,证实自噬性SNARE蛋白S...     

Protein complex expression by using multigene baculoviral vectors

Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression. Here we describe new transfer vectors and a combination of DNA recombination-based methods, which further facilitate the generation of multigene cassettes for protein coexpression (Fig. 1), thus providing a flexible platform for generation of protein expression vectors and their rapid regeneration for revised expression studies. Genes encoding components of a multiprotein complex are inserted into a suite of compatible transfer vectors by homologous recombination. These progenitor constructs are then rapidly joined in the desired combination by Cre-loxP-mediated in vitro plasmid fusion. Protocols for integration of the resulting multigene expression cassettes into the MultiBac baculoviral genome are provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored for this purpose. Detailed guidelines for multigene virus generation and amplification, cell culture maintenance and protein production are provided, together with data illustrating the simplicity and remarkable robustness of the present method for multiprotein expression using a composite MultiBac baculoviral vector.     

Construction of expression vectors for protein production in Gluconobacter oxydans

The characteristic ability of Gluconobacter oxydans to incompletely oxidize numerous sugars, sugar acids, polyols, and alcohols has been exploited in several biotechnological processes, for example vitamin C production. The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative proteins and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp. These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding β-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2 carrying the intrinsic lac promoter) promoters were identified.     

Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks

Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics.     

艰难梭菌毒素A原核表达及重组蛋白纯化

目的构建艰难梭菌(Clostridium difficile,C.difficile)毒素A羧基末端原核表达载体,优化诱导表达条件及纯化重组蛋白。方法利用聚合酶链式反应扩增C.difficile毒素A羧基末端基因序列,并将此序列转入pET-28b载体,构建pET-28b-tcdA重组表达载体,并将表达载体转化到BL21(DE3)感受态大肠埃希菌细胞中,分别在不同条件下进行诱导表达,获得最佳诱导表达条件后进行大量表达,最后用Ni柱对重组蛋白进行亲和纯化,得到纯化后的重组蛋白。结果成功构建了pET-28b-tcdA重组表达载体,其重组蛋白表达最佳诱导条件:菌液吸光度值取0.6、温度取25℃、IPTG终浓度取1.0mmol/L、诱导时间取10h。蛋白纯化咪唑磷酸盐洗脱液最佳浓度取200mmol/L。结论成功构建pET-28b-tcdA重组表达载体,在大肠埃希菌BL21(DE3)中可以有效表达,并获得高浓度重组蛋白,为进一步制备TcdA适配子奠定了一定实验室基础。     

Vectors for enhanced gene expression and protein purification in Salmonella

Improved expression vectors have been tested for protein expression studies in Salmonella spp. They are derived from the broad host range expression vector pNSGroE [Seleem, M.N., Vemulapalli, R., Boyle, S.M., Schurig, G.G. and Sriranganathan, N., 2004. Improved expression vector for Brucella species. Biotechniques 37, 740-744] and have several advantages (i) small in size (less than 3 kb); (ii) possess eleven unique restriction site to facilitate directional cloning; (iii) express proteins as His-tagged fusions for easy detection and purification; (iv) carry different promoters for various level of expression and (v) carry an UP element and RNA stem-loop for enhanced gene expression. We have demonstrated the ability of the new vectors to stably express heterologous proteins in Salmonella. We also demonstrated the utility of our vectors by detecting expression and purification of recombinant GFP. Our results suggest that these new vectors should improve gene expression in Salmonella, particularly those aimed at foreign antigen delivery.     

Attenuated protein expression vectors for use in siRNA rescue experiments

Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity, and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.     

Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.