Variety Of Expression Vectors Research Articles

Synthesizing and Expressing Native Ion Channels.

Synthesizing and expressing ion channels in heterologous systems enable the characterization of the functional properties of these proteins. The cDNA that encodes ion channels can be amplified directly from mRNA or synthesized de novo in its entirety before cloning into an appropriate expression vector. Gibson assembly is a powerful tool that allows rapid cloning and integration of protein-coding cDNA into a variety of expression vectors. Here we describe a method in which the cDNA encoding a native snake ion channel (NaV 1.4) is synthesized in four equal-sized pieces (or blocks), and then assembled and ligated into an expression vector. Once in an appropriate expression vector, the assembled cDNA can be used for synthesis of mRNA, and the mRNA injected and expressed in Xenopus oocytes. This method has significant advantages over traditional rtPCR and ligation-based cloning including speed, cost, ease of codon optimization, and inclusion of silent restriction sites for Gibson-based mutagenesis.

An Efficient Transient Expression System for Enhancing the Generation of Monoclonal Antibodies in 293 Suspension Cells.

Recombinant monoclonal antibodies (mAbs) are useful in research, diagnosis, and therapy. The increased demands of recombinant mAbs require efficient production systems. A variety of expression vectors have been developed for stable or transient production of mAbs in mammalian cells. Although a few commercial expression systems of mAbs can be listed, the high expense often impedes academic research. In this study, we described the development of a transient mammalian system based on a bicistronic vector, which contained an internal ribosome entry site (IRES) and enhancer elements to express the IgG1 light chain (LC) and heavy chain (HC) in one transcript. Optimization of all components of the expression system, including gene transfer methods and regulatory elements, yielded maximal expression levels in serum-free 293 suspension cells (Freestyle 293-F). This method enabled consistent production of the anti-programmed cell death protein 1 (PD-1) mAb up to 300 mg/L in less than one week under transiently transfected conditions. Furthermore, purified anti-PD-1 IgGs showed specific affinity to the target antigen human PD-1 and PHA-stimulated human T cells. The simplicity of the procedure made it suitable for the fast and high-yield production of IgG antibodies in small scales, which expedited the screening of a large number of recombinant candidates.

Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli.

BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study.ResultsWe firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers.ConclusionsIn this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0268-7) contains supplementary material, which is available to authorized users.

Comparison of Expression Vectors for Transient Expression of Recombinant Proteins in Plants

Production of recombinant proteins in plants is of increasing importance for practical applications. However, the production of stable transformed transgenic plants is a lengthy procedure. Transient expression, on the other hand, can deliver recombinant proteins within a week, and many viral vectors have been constructed for that purpose. Each of them is reported to be highly efficient, robust and cost-effective. Here, a variety of expression vectors which were designed for transient and stable plant transformation, including pPZP3425, pPZP5025, pPZPTRBO, pJLTRBO, pEAQ-HT and pBY030-2R, was compared for the expression of green fluorescent protein and β-glucuronidase in Nicotiana benthamiana by Agrobacterium-mediated transient expression. Our results show that pPZPTRBO, pJLTRBO and pEAQ-HT had comparable expression levels without co-infiltration of a RNA-silencing inhibitor. The other vectors, including the non-viral vectors pPZP5025 and pPZP3425, needed co-infiltration of the RNA-silencing inhibitor P19 to give good expression levels.Electronic supplementary materialThe online version of this article (doi:10.1007/s11105-013-0614-z) contains supplementary material, which is available to authorized users.

A Versatile and Efficient High-Throughput Cloning Tool for Structural Biology

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.

Advances in the Development of Genetic Tools for the Genus Rhodococcus

Rhodococci are robust biocatalysts and they show diverse capabilities with regard to the degradation of xenobiotic compounds including recalcitrant organic compounds such as heterocyclic aromatic compounds and polychlorinated biphenyls (PCBs). Furthermore, these microbes exhibit a broad range of enzymes that can produce valuable chemicals such as intermediates that have a medical importance. These distinct characteristics have encouraged researchers to use rhodococci for industrial, environmental, and pharmaceutical applications. Genome projects of several Rhodococcus species have revealed the presence of large plasmids that encode genes related to their specific abilities. Recent advances in the development of genetic tools along with the rapid progress in genome annotations will pave the way for the efficient cell engineering of rhodococci. Particularly, a variety of expression vectors and transposon mutagenesis systems will act as important tools, and the methods for gene expression analysis will become powerful tools for the discovery of desirable genes in cell engineering.

Bacterial expression of larval peritrophins of Chryosomya bezziana

Three candidate antigens, Chrysomya bezziana peritrophin-48, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42, were prepared for recombinant protein production in Escherichia coli using a variety of expression vectors. Cb peritrophin-48 was expressed as a recombinant protein possessing a carboxy-terminal hexaHis tag. Cb peritrophin-15 was expressed as both a glutathione S-transferase fusion protein and as an amino-terminal hexaHis tagged protein. The glutathione Stransferase Cb peritrophin-15 construct produced a heterogeneous group of fusion proteins. Cb peritrophin-42 was also expressed as an amino-terminal hexaHis tagged protein. The two putative domains of Cb peritrophin-42 were also separately expressed, again with amino-terminal hexaHis tags. Cultures of the hexaHis constructs Cb peritrophin-48, -15 and –42 were demonstrated to be useful for the production and purification of these protein antigens and were scaled-up for vaccine trials and protein characterization studies. Key words: Chrysomya , peritrophic membrane, peritrophin, recombinant protein, hexaHis tag, GST

Stable expression of nucleocapsid proteins of Puumala and Hantaan virus in mammalian cells.

The development of an in vitro-system for the stable expression and the analysis of native hantavirus proteins using hantaviral cDNA is of particular interest. As a first step the expression of the hantavirus nucleocapsid (N) proteins in mammalian cells was studied in more detail. The cDNA of the S-RNA segment of Puumala virus strain CG-1820 and Hantaan virus strain 76-118 was used for the construction of eucaryotic expression vectors that allow the generation and selection of mammalian cells harboring and expressing the N protein genes of hantaviruses. A variety of conventional and novel expression vectors as well as different mammalian cell lines were screened. The expression of the N protein of Puumala virus using the pGRE5-1 vector in which the transcription is under control of inducible glucocorticoid responsive elements (GRE) revealed that the Puumala virus N protein can be expressed in Vero E6 cells efficiently without any detectable cell toxicity. From the variety of expression vectors tested, it was found that pCR3.1 is the vector of choice for stable expression of hantavirus N proteins. The successful establishment of different mammalian cell lines expressing considerable amounts of Puumala and Hantaan virus N protein indicates that the stable and efficient expression of this particular viral protein in the cell lines of three evolutionary distinct species (human, monkey, and mouse) is possible. The system described here represents the experimental basis for further studies of hantavirus infection, replication, and pathogenesis using a reverse genetics approach.

Cytochromes P450 expression systems.

Catalytically active cytochrome P450 enzymes have been successfully expressed in bacterial, yeast, and mammalian cells. A variety of expression vectors have been used, resulting in both transient and stable expression. The system of choice depends on the goals of a particular project. Factors such as expense, ease of use, and yields required should govern the decision whether to use bacterial, yeast, insect, or mammalian cDNA expression. High-level expression of mammalian P450s in bacteria usually requires modifications of the amino-terminal region of the enzyme. The Escherichia coli P450-OR fusion proteins may also come of age for use in fermentation-production processes for the chemical industry. Many cytochromes P450 have been expressed in yeast, with variable levels of expression. Baculovirus, albeit somewhat tedious in having to individualize expression conditions, can produce high levels of enzyme. The standard mammalian cell expression systems, both transient and stable, have been of tremendous value to drug metabolism and carcinogenesis research and will continue to play a role in these areas.

Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major.

The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) of Leishmania major has been cloned and expressed in Escherichia coli and Saccharomyces cerevisiae. The strategy involved placing the entire 1560-bp coding sequence into a parent cloning plasmid that was designed to permit introduction of unique restriction sites at the 5'- and 3'-ends. In this manner, the entire coding sequence could be easily subcloned into a variety of expression vectors. High levels of TS-DHFR gene expression were driven by tac, pL and T7 RNA pol promoters in E. coli, and the GAPDH-ADH-2 promoter in S. cerevisiae. L. major TS-DHFR also complemented TS deficiency in E. coli. In E. coli, the protein accumulated to very high levels, but most was present as inactive inclusion bodies. Nevertheless, substantial amounts were soluble; up to 2% of the soluble protein was catalytically active TS-DHFR. In the yeast systems, essentially all of the bifunctional protein was soluble and catalytically active, and crude extracts contained about 100-fold more enzyme than do extracts from wild-type L. major. The expressed TS-DHFR from yeast and E. coli was purified to homogeneity by methotrexate-Sepharose affinity chromatography. About 8.5 mg of homogeneous, catalytically active protein is obtained from a 1-L culture of yeast, and 1.5 mg was obtained from 1 L of E. coli culture. A 200-L fermentation of the yeast expression system yielded a crude extract containing over 4 g of TS-DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)