Cytoplasmic Expression System Research Articles

1134. An Enhanced Autogene-Based Dual Promoter Cytoplasmic Expression System Yields Increased Gene Expression

1134. An Enhanced Autogene-Based Dual Promoter Cytoplasmic Expression System Yields Increased Gene Expression

Effects of codon usage versus putative 5 ′-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm

Matching the codon usage of recombinant genes to that of the expression host is a common strategy for increasing the expression of heterologous proteins in bacteria. However, while developing a cytoplasmic expression system for Fusarium solani cutinase in Escherichia coli, we found that altering codons to those preferred by E. coli led to significantly lower expression compared to the wild-type fungal gene, despite the presence of several rare E. coli codons in the fungal sequence. On the other hand, expression in the E. coli periplasm using a bacterial PhoA leader sequence resulted in high levels of expression for both the E. coli optimized and wild-type constructs. Sequence swapping experiments as well as calculations of predicted mRNA secondary structure provided support for the hypothesis that differential cytoplasmic expression of the E. coli optimized versus wild-type cutinase genes is due to differences in 5 ′ mRNA secondary structures. In particular, our results indicate that increased stability of 5 ′ mRNA secondary structures in the E. coli optimized transcript prevents efficient translation initiation in the absence of the phoA leader sequence. These results underscore the idea that potential 5 ′ mRNA secondary structures should be considered along with codon usage when designing a synthetic gene for high level expression in E. coli.

236. The Development and Characterization of a Cytoplasmic Expression System Based on the T7 Phage RNA Polymerase Protein

236. The Development and Characterization of a Cytoplasmic Expression System Based on the T7 Phage RNA Polymerase Protein

Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells

AbstractDesigning effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP+ cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34+ progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A–specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.

Cytoplasmic Expression of Ribozyme In Zebrafish Using a T7 Autogene System

A cytoplasmic ribozyme expression system, based on codelivery of a ribozyme vector, a T7 autogene vector, and T7 RNA polymerase (RNAP), has been developed and used to generate a specific phenotype in zebrafish by targeting a no tail (ntl) mRNA. The expression of the no tail ribozyme sequence is under the control of a tandem of two promoters: The T7 promoter and an adenoviral va 1 (pol III) promoter. The coinjection of the ribozyme vector pT7vaRz, the T7 autogene vector pT7T7, and the T7 RNAP resulted in rapid synthesis of the ribozyme against the ntl mRNA in the cytoplasm of the injected zebrafish embryos, generating no tail phenotypes in up to 10-20% of the injected embryos. The phenotypic change rates have been found to be related to the concentrations of the plasmid vectors and T7 RNAP injected and to the ratios of the three injected components. This cytoplasmic ribozyme expression system may be useful for efficiently targeting other mRNA and for various biomedical applications. These potential applications may include rapid identification of biological functions of novel genes from zebrafish and humans based on partial gene sequence information and gene therapy of genetic and acquired diseases.

A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes.

Inefficient nuclear transport of plasmid DNA continues to be a problem in nonviral vector-mediated gene transfer. This has made the cytoplasmic expression system an increasingly attractive idea. We have developed a new T7 RNA polymerase autogene for cytoplasmic expression containing both a CMV and a T7 promoter. The pCMV/T7-T7pol autogene does not encounter the problems associated with previously used autogenes. For instance, pCMV/T7-T7pol is easily amplified and purified from bacteria. Furthermore, the CMV promoter is used to drive the first round of synthesis of T7 RNA polymerase, thus negating the use of purified enzyme in the transfection complex. The endogenous T7 RNA polymerase produced from the CMV promoter could then act on the T7 promoter of pCMV/T7-T7pol in an autoregulatory mechanism. pCMV/T7-T7pol induces higher, more sustained levels (> 7 days) of reporter gene expression than that observed with the previously used autogene pT7 AUTO 2C- or with the nuclear expression system pCMV-CAT. This seems to be due to the high levels of T7 RNA polymerase protein that are detected in cells transfected with pCMV/T7-T7pol. This vector also functions as an efficient autogene since at least 50 times more mRNA is transcribed from the cytoplasmic T7 promoter as compared with the nuclear CMV promoter in pCMV/T7-T7pol. Therefore, pCMV/T7-T7pol could replace existing autogenes for regeneration of T7 RNA polymerase and efficient target gene expression.

High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the bacteriophage lambda head protein D.

The bacteriophage Lambda head protein D (gpD) is a small major capsid protein (110aa; 11.6kDa; pI=5.68, devoid of cysteine residues) that is essential for stable head morphogenesis. We found that a His-tagged derivative of gpD (gpHD) is a monomeric protein with efficient expression properties and high resistance towards thermally induced irreversible aggregation. In addition, gpHD can be used as a fusion partner for high-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli. Its broad utility is illustrated by the production of various mammalian proteins by fusion to its C-terminus. As a fusion partner, gpHD is thought to mediate optimal translation initiation while reducing inclusion body formation and protein degradation. In addition, it provides a His-tag for simple purification. gpHD may act as a 'cytoplasmic anchor' by keeping its unfolded fusion partner in solution, thereby providing more time for proper folding. An ever-increasing number of open reading frames (ORFs) are being identified in the various genome sequencing programs. gpHD has the potential to be harnessed for the development of highly efficient cytoplasmic expression systems that might contribute to the production and characterization of these novel polypeptides. Protein D is also an established fusion partner for phage display. It thus presents the attractive opportunity of coupling the selection of heterologous proteins from a phage library to their subsequent high-level expression.

Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase Hybrid System

AbstractThis unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (pT7). Using liposome‐mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7‐3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large‐scale work, protocols are provided for insertion of the pT7‐regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7‐3 into cells grown in suspension or for direct transfection into OST7‐1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse‐labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.

Gene Expression Using the Vaccinia Virus/ T7 RNA Polymerase Hybrid System

This unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (p(T7)). Using liposome-mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large-scale work, protocols are provided for insertion of the p(T7)-regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7-3 into cells grown in suspension or for direct transfection into OST7-1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse-labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.

Improved gene delivery formulations and expression systems for enhanced transaction efficiency

Abstract

Ｔ７ ＲＮＡ ｐｏｌｙｍｅｒａｓｅを利用した細胞質内遺伝子発現系の最適化

It is necessary to develop a more efficient gene expression system for gene therapy. A plasmid DNA, using eukaryoic or mammalian promoters, requires to localize into nuclear for gene expression. However, it is difficult to entry into nuclear, because nuclear pore size is not sufficient against the size of plasmid DNA. In this study, to develop a novel cytoplasmic gene expression system that dose not require nuclear localization of plasmid DNA to transcription, we examined the characterization of T7 cytoplasmic gene expression system. When co-transfected with pT7-IRES-L(luciferase expression plasmid containing T7 promoter) and T7 RNA polymerase into LLCMK2 cells, the gene expression of pT7-IRES-L was observed rapidly within 6hr after transfection and significant level of luciferase activity was detected. In contrast, pRSVL, a common plasmid DNA consist of luciferase expression plasmid and Rous sarcoma virus promoter, required 24-48hr for induction of gene expression. The gene expression level of the T7 system was enhanced with an increase in the amount of T7 RNA polymerase. To increase and prolong the gene expression, a plasmid DNA(pT7 AUTO-2) which contained the T7 RNA polymerase gene driven by the T7 promoter was co-transfected with pT7-IRES-L and T7 RNA polymerase. The plasmid DNA(pT7 AUTO-2) dose-dependently enhanced the luciferase gene expression by pT7-IRES-L and T7 RNA polymerase. In addition, we attempted to optimize the cytoplasmic gene expression system. The optimal ratio for co-transfection of pT7-IRES-L and pT7 AUTO-2 was 1 to 3 (mole ratio). These results suggest that T7 gene expression system may be useful in many gene therapies where transient but rapid efficient gene expression is required.

Cytoplasmic Gene Expression System Enhances the Efficiency of Cationic Liposome-Mediatedin VivoGene Transfer into Mouse Brain

Development of methodologies for gene transfer into the central nervous system (CNS) is important for fundamental research as well as clinical studies for gene therapy. Cationic liposomes (CL) are attractive vectors because of their safety and ease of use. However, to date only low rates of success have been reported. We succeeded in obtaining high transfection efficiencies into the newborn mouse brain in vivo by CL and a cytoplasmic gene expression system based on T7 RNA polymerase and T7 RNA polymerase- and the luciferase-gene with the T7 promoter sequence. This system showed an efficiency rate 2 orders of magnitude higher than the standard system, which used CL and luciferase genes with a Rous sarcoma virus promoter, pRSVL. In addition, in vitro experiments using LLCMK2cells showed that cytoplasmic gene expression occurred rapidly (within 6 h) after transfection. In contrast, pRSVL required 24-48 h for induction of luciferase expression. Our results suggest that the cytoplasmic gene expression system is useful for gene delivery into the CNS.

Delivery of a PCR amplified DNA fragment into cells: a model for using synthetic genes for gene therapy.

Synthetic genes offer many potential advantages over conventional plasmid DNA, such as simplicity in purification, absence of endotoxin contamination, and more importantly, flexibility in chemical modifications to render them specific properties. We have used PCR amplified fragments as a model to test the feasibility of using synthetic genes for gene therapy. The CAT reporter gene driven by the CMV promoter (CMV-CAT), ie a nuclear expression system, or by the bacteriophage T7 promoter (T7-CAT), ie a cytoplasmic expression system, was used to evaluate this concept. The expression efficiency of both plasmids (pUCCMV-CAT and pT7-CAT) and their corresponding PCR fragments (fCMV-CAT and fT7-CAT) were compared on a molar basis. Limited expression of CAT was found with fCMV-CAT. However, fT7-CAT consistently gave a CAT activity comparable to that of pT7-CAT. When fT7-CAT was codelivered with pCMV/T7-T7pol (a self-amplifying-T7 RNA polymerase autogene), high CAT activity could be detected up to 9 days. This expression was much longer than the duration of expression with a nuclear expression system. These encouraging results imply that gene therapy with synthetic genes could be both feasible and efficient.

DC-Chol lipid system in gene transfer

Lipidic systems including cationic liposomes offer many potential advantages for delivering functional DNA to cells in intact animals. DC-Chol:DOPE, a cationic liposome formulation developed in this laboratory, is highly efficient for delivering nucleic acids into various cell types in vitro as well as in vivo. Mixing DNA with cationic liposomes produce condensed DNA along with tubular structures and aggregated liposomes. Interaction with cell membrane, followed by endocytosis and disruption of endosomes, appears to be the main mechanism of cytoplasmic delivery of DNA by DNA/cationic liposome complex. In an attempt to overcome the low efficiency of nuclear entry of cytoplasmic DNA, a limiting step for the overall expression level of the transgene, a cytoplasmic expression system was developed. A plasmid containing the reporter gene, chloramphenicol acetyltransferase (CAT), driven by the bacteriophage T7 promoter, following co-delivery with T7 RNA polymerase by DC-Chol cationic liposomes into cells, gives a rapid, but transient CAT gene expression. However, strong and sustained gene expression could be achieved by co-delivery of a T7 RNA polymerase enzyme regenerating system such as a T7 autogene. An independent study found that a single i.p. injection of cisplatin could sensitize lipofection of tumors in situ. A combined and sequential protocol was therefore proposed for cancer gene therapy.

The Translational Repression Mediated by the Platelet-derived Growth Factor 2/c-sis mRNA Leader Is Relieved during Megakaryocytic Differentiation

Expression of the platelet-derived growth factor 2/c-sis gene is highly restricted and controlled at multiple levels. Its structured mRNA leader, which is unusually long (1022 nucleotides), serves as a potent translational inhibitor. One of the sites of PDGF2 synthesis is megakaryocytes, implying that PDGF2 translation efficiency is modulated during megakaryocytic differentiation. To study the role of the mRNA leader as a translational cis-modulator, the hybrid T7/vaccinia cytoplasmic expression system was used to disconnect between determinants controlling transcription, alternative splicing, and mRNA stability from those controlling translation. Chimeric transcripts in which the human PDGF2/c-sis mRNA leader positioned in frame upstream of a reporter gene were used to determine whether the mRNA leader can confer variable translational efficiencies during differentiation. It is demonstrated that there is a time window during megakaryocytic differentiation of K562 cells in which the strong translational inhibition by PDGF2/c-sis mRNA leader is relieved. The time course of the translational repression relief is similar to that of PDGF2/c-sis transcriptional induction during the differentiation process. A 179-nucleotides CG-rich fragment immediately upstream of the initiator AUG codon is necessary for coffering stringent modulation of the translational efficiency. In NIH3T3 overexpressing translation initiation factor eIF4E, the inhibitory effect of the mRNA leader of c-sis is not relieved, suggesting that the changes in the translational machinery during megakaryocytic differentiation are beyond eIF4E activity. The possible involvement of a 5'-end-independent translational mechanism is discussed.

Gene Therapy Using Dc-Chol Liposomes

AbstractGene therapy offers the potential to deliver genetic material inside the cells for correcting a disease phenotype. Various techniques for the transgene delivery have been developed, among which cationic liposome mediated delivery is widely acceptable. DC-Chol/DOPE cationic liposomes, synthesized in our laboratory, have several alluring features such as low toxicity, non-immunogenicity, ease of utilization and stability. These liposomes have been successfully used as a vector in clinical trials for treating melanoma and cystic fibrosis. Only a small portion of the DNA-liposome complex taken up by the cell is eventually available for gene expression in the nucleus. To overcome the problem of limited expression, we have developed a cytoplasmic expression system in which the reporter gene is placed under the control of a bacteriophage T7 promoter. To initiate the expression of the gene either purified T7 RNA polymerase or T7 autogene expression vector is transfected along with the reporter gene. Prolo...

A Sustained, Cytoplasmic Transgene Expression System Delivered by Cationic Liposomes

A plasmid containing the reporter gene, chloramphenicol acetyltransferase (CAT), driven by the bacteriophage T7 promoter was co-delivered with purified T7 RNA polymerase by the DC-chol cationic liposomes into human embryonic kidney 293 cells to obtain a transient (2 days) CAT gene expression. To prolong the expression, a T7 autogene which contains the T7 RNA polymerase gene driven by the T7 promoter was included in the transfection complex as a self-amplifying regeneration mechanism for the polymerase. High level CAT gene expression was observed up to 5 days after transfection. This strong and sustained expression system should be useful in gene transfer experiments.

A self-initiating eukaryotic transient gene expression system based on contransfection of bacteriophage T7 RNA polymerase and DNA vectors containing a T7 autogene.

A novel cytoplasmic gene expression system has been developed. This system differs from other expression systems in that it relies on the co-delivery of plasmid DNA and T7 RNA polymerase (RNAP) during transfection. The plasmid contains a T7 RNAP gene driven by the T7 promoter (T7 autogene) and a functional/reporter gene driven by another T7 promoter (T7T7/T7-gene construct). Once this DNA-enzyme complex is introduced into eukaryotic cells, the transcription of the T7 RNAP and the functional/reporter genes is initiated by the co-delivered T7 RNAP. The T7 RNAP, which is responsible for the initiation and maintenance of expression of both T7 and functional/reporter genes, is replenished by translation of newly synthesized T7 mRNA. This T7 system was designed in such a manner that the expression of the functional/reporter genes can occur in the cytoplasm and does not require any nuclear involvement. When transfected by either a pT7T7/T7Luc or a pT7T7/T7hGH plasmids with the cointroduced T7 RNAP, mouse L cells were found to express high levels of luciferase immediately after transfection, apparently due to the cytoplasmic gene expression; the expression of human growth hormone (hGH) could be sustained for at least 6 days. Both T7 and hGH mRNA were expressed by the cells transfected with pT7T7/T7hGH. These results suggest that this cytoplasmic expression system may be used for certain targets of somatic gene therapy.

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope.

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202-221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site.

Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.

A mouse cell line that constitutively synthesizes the bacteriophage T7 RNA polymerase was constructed. Fluorescence microscopy indicated that the T7 RNA polymerase was present in the cytoplasmic compartment. The system provided, therefore, a unique opportunity to study structural elements of mRNA that affect stability and translation. The in vivo activity of the bacteriophage polymerase was demonstrated by transfection of a plasmid containing the chloramphenicol acetyltransferase (CAT) gene flanked by T7 promoter and termination signals. Synthesis of CAT was dependent on the presence of a cDNA copy of the untranslated region of encephalomyocarditis virus (ECMV) RNA downstream of the T7 promoter, consistent with the absence of RNA-capping activity in the cytoplasm. CAT expression from a plasmid, pT7EMCAT, containing the T7 and EMCV regulatory elements was detected within 4 hr after transfection and increased during the next 20 hr, exceeding that obtained by transfection of a plasmid with the CAT gene attached to a retrovirus promoter and enhancer. Nevertheless, the presumably cap-independent transient expression of CAT from pT7EMCAT was increased more than 500-fold when the transfected cells also were infected with wild-type vaccinia virus. A protocol for high-level expression involved the infection of the T7 RNA polymerase cell line with a single recombinant vaccinia virus containing the target gene regulated by a T7 promoter and EMCV untranslated region.