Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda

Constitutive and thermoinducible expression plasmids based on strong P(R),P(L) promoters from phage lambda were compared for production of TNF-alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF-alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2-3-fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible.     

pHUSH: a single vector system for conditional gene expression

Background

Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector.

Results

Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer.

Conclusion

We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.     

New phytoviral vector for superexpression of target proteins in plants

Previously, we obtained a new viral vector based on the Alternanthera mosaic virus strain MU (AltMV-MU). The gene of interest was placed under control of two subgenomic promoters (sgp). Viral vector provided the superexpression of target proteins in Nicotiana benthamiana. In the present work, to increase the level of protein expression, this viral vector was modified and the coat protein gene was placed under control of three sgp. The new viral vector provided superexpression of the protein of interest in plants, and its amount was more than 50% of the total soluble protein in cells. The efficiency of the target protein expression in the plants transformed with viral vectors containing two and three sgp was compared.     

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.     

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.     

Enhanced expression of cytochrome P450s from lac-based plasmids using lactose as the inducer

The cytochrome P450 expression systems used in Escherichia coli are highly regulated and involve the use of the lac repressor to control expression. Induction in these systems utilizes the nonmetabolizable analog of lactose, isopropyl-beta-D-thiogalactopyranoside (IPTG), which is the most expensive compound required for an E. coli expression system. To determine if the natural inducer lactose could be used to induce cytochrome P450 expression we examined the expression of three P450 enzymes in E. coli using two different expression systems, pTrc99A and the T7-based PET22b vector. For both systems lactose was found to induce expression of active P450 to concentrations that exceeded the levels achieved with IPTG. A 20-liter fermentation of a P450 expression system in the pTrc plasmid in which lactose was used as the inducer resulted in 2.4 micromol P450/liter, with a total yield of 2 g of cytochrome P450. The use of lactose for protein expression in E. coli should be broadly useful for the inexpensive, large-scale production of heterologous proteins in E. coli.     

A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.     

志贺毒素B（ShT-B）亚单位在两种表达载体中表达形式分析

用KpnⅠ和HindⅢ双酶切pGEMTB3克隆质粒,得到大小约为225bp的ShTB基因片段,分别将其插入到经双酶切的pQE40和pQE30表达载体中,构建了2个ShTB的重组表达质粒pQE40B3和pQE30B2,分别转化到E.coliM15,经IPTG诱导后,重组质粒目的蛋白均得到表达。其中,pQE40B3表达蛋白约占菌体总蛋白的37%,主要为包涵体形式。pQE30B2表达蛋白约占菌体总蛋白的16%,主要为可溶性形式,约9.2%。为重组抗原的制备提供了必要的物质基础。     

Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability

A series of plasmid-based expression vectors have been constructed allowing stable intracellular expression of recombinant proteins in Bacillus subtilis strains. These expression vectors are based on the recently described Escherichia coli-B. subtilis shuttle vector pMTLBS72 which replicates as theta circles. Besides the weak constitutive promoter P(lepA), we inserted three different controllable promoters: P(gsiB) which can be induced by heat and acid shock, and by ethanol, P(xylA) and P(spac) which respond to the addition of xylose and IPTG, respectively. The versatility of these expression vectors was demonstrated by fusing their promoters to a reporter gene and by overexpression of the HtpG protein with three of them. All recombinant vectors exhibited full structural stability.     

Tightly regulated, beta-estradiol dose-dependent expression system for yeast

We have refined the regulated expression of UASGAL1, 10-driven genes in yeast by modifying a vector encoding the beta-estradiol inducible activator, GAL4.ER.VP16 (GEV). The expression of GEV was placed under the regulation of the low-level, constitutive MRP7 promoter, and beta-estradiol-regulated expression was monitored by the expression of an integrated UASGAL10-lacZ reporter and by immunoblot analysis of a UASGAL1-regulated gene product. Target gene expression regulated by low levels of GEV has several advantages over the standard galactose-inducible expression systems. (i) Most importantly, the target gene expression is undetectable in the absence of hormone; (ii) target gene expression is beta-estradiol dose-dependent, and variable levels of target gene expression from low to several hundred-fold induction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4 delta strains. In addition, any UASGAL1,10 expression construct can be used without modification of the target gene or many gal4 delta host strains, and GEV vectors are compatible with other inducible yeast expression systems. This method may be useful to researchers investigating the functions of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.     

Lactococcus lactis,an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

Background

Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.

Methodology/Principal Findings

The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.

Conclusions/Significance

Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.     

Development of a New Generation of Vectors for Gene Expression,Gene Replacement,and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.     

pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

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.     

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

The human cytomegalovirus and elongation factor 1?? promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines.