Efficient Expression Vector Research Articles

Identification and analysis of putative promoter motifs in Flavivirus genome

The genus Flavivirus comprises medically significant pathogenic virus; causing several infections in humans worldwide. Flavivirus genomes are 10-11 kb approximately and encode both structural and non structural region. The non structural region plays fundamental role in the stability, regulation and cell cycle of virus. The complete genomes of 26 Flavivirus were used for identification of promoter motifs through in silico approaches. The promoter sequences were encoded in merely 16 viruses and 10 viruses could not encode it. All these in silico identified promoter motifs was confirmed and verified with the known experimental data. This analysis suggests that presence of promoter may play a crucial role in the pattern of gene expression, regulation networks, cell specificity and development. It may also be useful for designing efficient expression vector and target specific delivery system in the gene therapy.

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

Expression of modified VHb gene and fused insectidal gene in tobacco

AbstractThe highly efficient plant binary expression vector pGBIF4ABCVHB was constructed using the artificially modified, synthesized VHb gene and fused insectidal gene (GFMcryIA and CPTI). Transgenic tobacco plants were obtained by Agrobacterium-mediated transformation. For 32 plants, the bivalent genes were inserted successfully into the tobacco genome and detected by polymerase chain reaction (PCR) amplification. Southern blot and Western blot analyses showed that VHb gene was expressed in the transgenic plants. Toxicity assay indicated that the fused insectidal gene expressed pesticidal toxin protein. The net weight of transgenic tobacco plants exceeded that of non-transgenic ones by 8%. Compared to non-transgenic tobacco plants, transgenic plants appeared to be high-yielding, insect-resistant varieties.

Complete In Vivo Reversal of the Multidrug Resistance Phenotype by Jet-injection of Anti-MDR1 Short Hairpin RNA-encoding Plasmid DNA

Triggering the RNA interference (RNAi) pathway by inducing the expression of short hairpin RNA (shRNA) molecules has become a promising tool for efficient silencing of a given gene in gene therapy applications. In this study, shRNA encoding DNA was utilized to reverse the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype in vivo. For the first time, the nonviral jet-injection technology was applied for delivering naked shRNA-vector constructs for direct intratumoral in vivo transfer. The highly efficient anti-MDR1 shRNA expression vectors were applied twice in the human MDR1/P-gp overexpressing MaTu/ADR cancer xenograft-bearing mice, and twice in the corresponding drug-sensitive parental MaTu tumor xenograft bearing mice as well. Two days after anti-MDR1 shRNA vector injection, the expression level of the MDR1 messenger RNA (mRNA) was decreased by more than 90% and the corresponding MDR1/P-gp protein was no longer detectable in the tumors. Two jet-injections of anti-MDR1 shRNA vectors into the tumors, combined with two intravenous (IV) administrations of doxorubicin, were sufficient to achieve complete reversal of the drug-resistant phenotype. The data show that jet-injection delivery of shRNA-expressing vectors is effective in reversing MDR1/P-gp-mediated MDR in vivo, and is therefore a promising strategy for making tumors with an MDR1/Pgp-dependent MDR phenotype revert to a drug-sensitive state.

Efficient Systemic Expression of Bioactive IL-15 in Mice upon Delivery of Optimized DNA Expression Plasmids

Efficient expression vectors for interleukin 15 (IL-15) were developed combining RNA/codon optimization and modification of the IL-15 native long signal peptide. These changes resulted in elevated cytoplasmic levels of the optimized mRNA and more than 100-fold improved production of secreted human IL-15 protein. Similar modifications have also led to greatly increased rhesus macaque and murine IL-15 production. Comparison of different heterologous secretory signals showed that the tissue plasminogen activator signal is most efficient for the production of extracellular IL-15. Upon intramuscular injection of the fully optimized expression vectors in mice, IL-15 was readily detected in the serum. Serum levels represented <1% of intramuscular IL-15 and were sufficient in causing some systemic effects, such as increasing the frequency of natural killer (NK) cells in the liver. Upon hydrodynamic DNA delivery in mice, very high levels of IL-15 were produced, which increased the frequency of NK cells in liver as well as in spleen and lung. These optimized expression vectors have potential applications in vaccine and immunotherapy approaches against AIDS and cancer.

A new Gateway ® vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis

A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism from which the proteins are derived. Here we describe methods for efficiently identifying M. tuberculosis proteins that express in soluble form in Mycobacterium smegmatis. We have adapted the M. smegmatis expression vector pYUB1049 to the Gateway ® cloning system by the addition of att recombination recognition sequences. The resulting vector, designated pDESTsmg, is compatible with our in-house Gateway ® methods for E. coli expression. A target can be subcloned into pDESTsmg by a simple LR reaction using an entry clone generated for E. coli expression, removing the need to design new primers and re-clone target DNA. Proteins are expressed by culturing the M. smegmatis strain mc 24517 in autoinduction media supplemented with Tween 80. The media used are the same as those used for expression of proteins in E. coli, simplifying and reducing the cost of the switch to an alternative host. The methods have been applied to a set of M. tuberculosis proteins that form inclusion bodies when expressed in E. coli. We found that five of eight of these previously insoluble proteins become soluble when expressed in M. smegmatis, demonstrating that this is an efficient salvage strategy.

Silencing of puroindoline a alters the kernel texture in transgenic bread wheat

Grain hardness is an important end-use quality parameter of bread wheat, and one of the most important characters for quality improvement. The objective of this study was to further understand the function of puroindolines and the underlying mechanism in the formation of kernel texture. The highly efficient expression vector pUBPa harboring puroindoline a ( Pina) was introduced into the bread wheat cultivar Zhongyou 9507-60 via biolistic transformation and transgenic plants were obtained. The integration of the foreign Pina gene was confirmed by PCR and genomic DNA Southern blot analysis. The levels of friabilin on the surface of water-washed starch granules varied among the transgenic lines. SDS-PAGE analysis of Triton X-114 extracted protein showed that the PINA protein was absent in three transgenic lines, indicating that the endogenous Pina gene most likely had been co-suppressed by the over-expression of the Pina transgene. SKCS kernel hardness and scanning electron microscopy analysis further confirmed the changes of kernel texture in these lines.

High level expression of His-tagged colicin 5 in E. coli and characterization of its narrow-spectrum bactericidal activity and pore-forming action

Since antibiotics with a broad spectrum of activity would select for resistance among the normal flora, colicins having a narrow spectrum of activity can potentially be developed as novel antibiotics. Colicin-based bactericidal proteins with modified spectra of activity might also be developed by further gene fusion or gene modification. To achieve these goals, it is necessary to first build an efficient system to produce large amounts of colicin. In the presence of an immunity gene, we successfully constructed an expression vector pQE30- cfa- cfi producing high levels of His-tagged colicin 5 (60–80 mg/L). We found that the purified His-tagged colicin 5 possesses narrow-spectrum bactericidal activity against nonimmune Escherichia coli cells. It is highly toxic to sensitive E. coli cells at a low concentration of 0.01 μg/ml, while it is nontoxic to other tested gram-negative bacteria, gram-positive bacteria and yeast at a high concentration of 1000 μg/ml. His-tagged colicin 5 kills sensitive cells by permeabilizing their cell membranes. It is not hemolytic to rabbit erythrocytes and has no obvious cytotoxicity to other nucleated mammalian cells at a high concentration of 500 μg/ml. The His-tagged colicin 5 is similar to wild-type colicin 5 in spectrum and bactericidal activity against E. coli. It is a potential novel antibiotic particularly for treating human and animal infections caused by pathogenic E. coli. Besides producing high level of colicin 5, the highly efficient expression vector constructed here might also be a useful tool to develop colicin-based artificial bactericidal proteins.

Construction of a Bicistronic Lentiviral Vector for Efficient Transduction and Expression of Multiple Therapeutic Genes in Adult Stem Cells

Adult stem cells (ASC) have emerged as a potentially useful substrate for neovascularization and repair of ischemic myocardium and injured blood vessels. Pseudotyped retroviral vectors efficiently transduce ASC, providing long-term stable expression of therapeutic genes. However, the cytotoxicity of these vectors is increased in conditions where multiple transductions may be required to simultaneously express more than one gene. In order to minimize the toxicity associated with multiple retroviral transductions we designed a bicistronic lentiviral vector for simultaneous expression of two genes. To construct the bicistronic vector we cloned a modified internal ribosome entry sequence (IRES) from pIRES2AcGFP1 between two reporter genes (Green Fluorescent protein, AcGFP1 and Firefly Luciferase, Luc). To test the strength of translation of the IRES we generated two complementary vectors, the first with GFP and Luc cloned 5′ and 3′ of the IRES sequence, respectively; the second vector with the reporter genes cloned in the reverse order with respect to the IRES. Luciferase activity was determined using the Bright-Glo Luciferase Assay System. The first construct was packaged into lentiviral particles by four plasmid transfection into HEK 293FT cells, using VSG-V for viral pseudotyping. Analyis of reporter gene expression in transduced HEK 293 cells showed that the bicistronic expresses both reporter genes with minimal attenuation of expression by the IRES sequence. Transduction efficiency of cells was consistently > 40% with no signs of cytotoxicity. Supported by grants from CIHR and HSFO to L.G. Melo and C.A. Ward

Late gene mutants of bacteriophage λ as an efficient expression vector

The disadvantage of a bacteriophage λ expression system is that the replicated λ DNA containing a foreign gene is coated by a phage head, and cell lysis occurs before sufficient expression of the cloned gene. The late genes of the bacteriophage λ system are related to λ DNA packaging and host cell lysis. This study investigated the late gene mutants of the bacteriophage λ in an attempt to develop an efficient expression vector system. The bacteriophage λNM1070, which is a W −, E − and S − mutant, had increased cell longevity due to the S − mutation. However, the genetic stability of this expression system was not high enough because the W − and E − mutation caused a defect in its re-infection. The bacteriophage λHL1, which is a Q − mutant, represses the transcription of all the late genes, even though this repression was not complete. This incomplete repression was rather beneficial to the cloned gene expression because it produced some matured phage particles, which re-infected the segregated host cell and increased the genetic stability. Therefore, a double mutant of the genes S and Q was constructed in order to exploit the merits of λNM1070 and λHL1. This λ Q − S − mutant had higher host cell longevity and cloned gene expression than the λNM1070 and λHL1.

Immunogenicity and cytoadherence of recombinant heparin binding haemagglutinin (HBHA) of Mycobacterium avium subsp. paratuberculosis: Functional promiscuity or a role in virulence?

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's Disease, a chronic granulomatous enteritis of ruminants. Recently, an association of MAP bacilli with Crohn's disease in humans has been proposed. Due to genetic similarities and serological cross-reactivity of the M. avium complex with other mycobacteria, functional analysis of species-specific proteins may allow new insights into the pathogenesis of mycobacterial diseases. We report production and molecular characterization of the recombinant HBHA from the MAP complex bacilli. The HBHA was expressed in Escherichia coli and Mycobacterium smegmatis using efficient expression vector systems. The recombinant HBHA was found to be immunogenic and therefore induced antibody responses in cattle against the MAP bacilli with a possible cross reactivity with M. bovis infection. The MAP complex HBHA was thus found to be a target of the host humoral responses in Johne's disease. The recombinant HBHA protein was also found to be adherent to the Caco2 cell lines in-vitro, a significant observation to understand possible virulence mechanisms. Since M. tuberculosis HBHA was earlier shown to be involved in dissemination of the tubercle bacilli, the immunogenicity and cytoadherent nature of this MAP protein possibly suggests functional promiscuity.

Design of Hammerhead Ribozymes that Cleave Murine Sry mRNA In Vitro and In Vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.

Long-term cardiac gene expression using a coxsackieviral vector

Efficient myocardial gene transfer in the intact adult heart is difficult using conventional transfer vectors. Since coxsackievirus B3 (CVB3) is cardiotropic, it may be possible to exploit its cardiotropic characteristics to design a vector for gene transfer to the intact heart. We generated a recombinant CVB3 cDNA by inserting a green fluorescent protein (GFP) gene immediately upstream from the VP0 capsid protein of CVB3. The infectious virus (rCVB3-GFP) was recovered from the supernatants of the transfected Cos-7 cells, and was grown in HeLa cells to titers of 10 11 pfu/ml. In the rCVB3-GFP infected HeLa cells and neonatal rat cardiac myocytes, GFP protein expression was documented by immunoblot and by fluorescent microscopy. GFP expression was maintained after five passages in HeLa cells. To test in vivo expression of GFP, we infected 8-week-old inbred female Balb/C mice with 10 6 pfu of rCVB3-GFP, intraperitoneally. GFP was present in up to 30% of cardiac myocytes over the 8 weeks post infection (p.i.) and it was co-localized with CVB3 infection. Surprisingly, in spite of detection of GFP up to at least 8 weeks after infection, there was no mortality in the mice. It is possible to express exogenous proteins in the intact heart after an intraperitoneal (i.p.) injection of recombinant coxsackievirus. The duration of expression persisted for at least 8 weeks with little immune response nor mortality. These results demonstrated that the cardiac tropism of CVB3 could be used to design vectors for efficient gene expression in the intact heart.

Baculovirus as a highly efficient expression vector in insect and mammalian cells.

Baculovirus has been widely used for the production of recombinant proteins in insect cells. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of baculovirus have been greatly expanded. The prospects and drawbacks of baculovirus‐mediated gene expression, either in insect or in mammalian cells, are reviewed. Recent progresses in expanding the applications to studies of gene regulation, viral vector preparation, in vivo and ex vivo gene therapy studies, generation of vaccine vectors, etc are discussed and the efforts directed towards overcoming the existing bottlenecks are particularly emphasized.

Development of real-time RT–PCR for evaluation of JEV clearance during purification of HPV type 16 L1 virus-like particles

Insect cell culture has greatly increased in part due to the widespread use of insect virus-based vectors for efficient expression of foreign proteins. Insect cells such as Sf9 cells are susceptible to arboviruses which may pose a safety concern by adventitious introduction during the production process. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived biotechnological products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses that are known to be infectious in insect cells. Here we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT–PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. The JEV specific primer was selected from the 3′ untranslated region, and the expected band size was 323 base pairs (bp). The sensitivity of the assay was calculated to be approximately 15 TCID 50per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. JEV clearance was determined during the purification process of rHPV-16 L1 VLPs by CsCl equilibrium density centrifugation. The comparative results obtained by real-time RT–PCR assay for JEV and infectivity titrations suggested that the real-time RT–PCR assay could have an additive effect on the interpretation and evaluation of virus clearance, especially during the virus removal process.

Dual-expression vectors for efficient protein expression in both E. coli and mammalian cells.

AbstractIn the near future, the nucleotide sequence of the genomes from many different organisms will be available. The next and more challenging step will be to characterize the biological role of each gene and the way in which the encoded protein functions in the cell. Dual-expression vectors for expression of proteins encoded by these genes in mammalian and bacterial cells can be used for this characterization. Typically, eukaryotic genes are expressed in mammalian cells to characterize biological functions and in bacterial cells to facilitate isolation of the protein. This generally requires the use of more than one vector. In contrast, use of a dual-expression vector eliminates the need to subclone from one vector system to another by combining the essential features of both eukaryotic and prokaryotic vectors in a single vector.KeywordscDNA InsertThrombin Cleavage SiteSupercompetent CellProkaryotic VectorOmpT ProteaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli.

Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

Retroviral vectors produced in the cytoplasmic vaccinia virus system transduce intron-containing genes.

Introns and polyadenylation (pA) sites are known to improve transcript stability and nuclear-cytoplasmic transport and are normally present in efficient gene expression vectors. Standard retroviral vectors, however, do not allow the inclusion of such sequence elements, as mRNA processing at internal splice and pA sites interferes with the production of functional full-length vector genomes. In this report we examined the capability of hybrid vaccinia/retroviral vectors to transduce complex gene cassettes with nuclear RNA processing signals within the retroviral genome. A retroviral vector was constructed that contains a gene of interest (the human coagulation factor IX [FIX] cDNA), including an intron and an internal pA site. The modified proviral vector genome was cloned downstream of a vaccinia virus promoter and was inserted into the vaccinia virus genome. Infection of a packaging cell line with the recombinant vaccinia virus vector resulted in secretion of retroviral particles at average titers of 10(5) CFU per ml of cell culture supernatant. Due to the cytoplasmic transcription and the nonrecognition of nuclear transcription signals in the vaccinia virus system, full-length transcripts were obtained that still contained the intron. In the retrovirally transduced cell lines the FIX transcripts were terminated at the internal pA site. The transcripts were quantitatively spliced, and FIX was secreted. Recombinant cell lines with stable single-copy inserts containing sequence elements necessary for efficient gene function could be generated. Thus, a relatively simple cytoplasmic system for the generation of complex retroviral vectors is described. Retroviral vectors transducing intron-containing gene cassettes may play a further role in gene therapy applications.

Engineering of a Sagiyama alphavirus RNA-based transient expression vector.

Sagiyama virus (SAGV), a strain of Getah virus in the genus Alphavirus in the family Togaviridae, has a broad host range in vertebrates and invertebrates but is not pathogenic for humans. We engineered the SAGV genome as an efficient transient expression vector using the full-length infectious cDNA clone pSAG2 as the background. A green fluorescent protein (GFP) gene was used as a reporter gene and expressed from a subgenomic mRNA. When the GFP gene was placed downstream of the intact capsid protein gene or an internally deleted capsid protein gene encoding the N-terminal 9 amino acids and C-terminal 149 amino acids, autoproteolysis occurred efficiently at the boundary site to release GFP from the N-terminally-fused capsid-protease domain. GFP was also expressed efficiently without the 5'-terminal region of the capsid protein gene, suggesting that SAGV capsid protein gene does not have a translation enhancer sequence. To provide structural proteins for pseudovirion formation, a nonviable mutant construct, pSAG2.3L, which contains a Gly-to-Leu substitution at the - 2 position of the nsP3/4 cleavage site, was used as a helper. GFP was expressed up to 50 pg from 1 X 10(6) BHK21 cells after inoculation of pseudovirions. The C6/36 mosquito cell was also a suitable host for a large scale expression of GFP using pseudovirions. In addition to high-level transient expression, safeness of SAGV should give an advantage over other alphavirus expression vectors.

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspensioncultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was discussed.