FMDV 2A Research Articles

Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas

BackgroundChlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae.ResultsC. reinhardtii was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide.ConclusionsThis work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.

Expression of Cholesterol Hydroxylase/Lyase System Proteins in Yeast S. cerevisiae Cells as a Self-Processing Polyprotein.

2A peptide discovered in Picornaviridae is capable of self-cleavage providing an opportunity to carry out synthesis of several proteins using one transcript. Dissociation in the 2A sequence during translation leads to the individual proteins formation. We constructed cDNA including genes of the bovine cholesterol hydroxylase/lyase (CHL) system proteins-cytochrome P450scc (CYP11A1), adrenodoxin (Adx) and adrenodoxin reductase (AdR), that are fused into a single ORF using FMDV 2A nucleotide sequences. The constructed vectors direct the expression of cDNA encoding polyprotein P450scc-2A-Adx-2A-AdR (CHL-2A) in Escherichia coli and Saccharomyces cerevisiae. The induced bacterial cells exhibit a high level of CHL-2A expression, but polyprotein is not cleaved at the FMDV sites. In yeast S. cerevisiae, the discrete proteins P450scc-2A, Adx-2A and AdR are expressed. Moreover, a significant proportion of AdR and Adx is present in a fusion Adx-2A-AdR. Thus, the first 2A linker provides an efficient cleavage of the polyprotein, while the second 2A linker demonstrates lower efficiency. Cholesterol hydroxylase/lyase activity registered in the recombinant yeast cell homogenate indicates that the catalytically active CHL system is present in these cells. Consequently, for the first time the mammalian system of cytochrome P450 has been successfully reconstructed in yeast cells through expressing the self-processing polyprotein.

A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii offers a great promise for large-scale production of multiple recombinant proteins of pharmaceutical and industrial interest. However, the nuclear-encoding transgenes usually are expressed at a low level, which severely hampers the use of this alga in molecular farming. In this study, the promoter of the endogenous intraflagellar transport 25 (IFT25) gene of C. reinhardtii was tested for its ability to drive the expression of green fluorescent protein (GFP), which functions as a readout for target gene expression. IFT25 promoter (IFT25P) alone was not able to drive GFP expression to a detectable level. IFT25P, however, can drive robust IFT25-GFP fusion protein expression when the intron-containing IFT25 gene was inserted between IFT25P and GFP cDNA. When an extended version of foot-and-mouth virus 2A protease (2AE) sequence was further inserted between the intron-containing IFT25 gene and the GFP cDNA, discrete GFP protein was observed to release from the IFT25-2AE-GFP polyprotein via 2A self-cleaving with a cleavage efficacy of approximately 99%. The monomer GFP was accumulated to a level of as high as 0.68% of total soluble proteins. To test whether the newly developed bicistronic IFT25P-IFT25-2AE expression system can be used to overexpress heterologous proteins of different origins and sizes, we inserted codon-optimized cDNAs encoding a Trichoderma reesei xylanase1 (25kDa) and a Lachnospiraceae bacterium ND2006 type V CRISPR-Cas protein LbCpf1 (147kDa) to the vector and found that the production of xylanase1 and LbCpf1 was as high as 0.69 and 0.49% of total soluble protein. Our result showed that IFT25P-IFT25-2AE system is more efficient to drive nuclear gene expression in C. reinhardtii than other conventionally used promoters, thus representing a novel efficient recombinant protein expression tool and has the potential to be scaled for commercial production of nuclear-encoded recombinant proteins of different sizes and origins in C. reinhardtii.

A Promising Trigene Recombinant Human Adenovirus Vaccine Against Classical Swine Fever Virus

Classical swine fever (CSF) vaccine based on HAdV-5 had achieved an efficient protection in swine. Both classical swine fever virus (CSFV) E0 glycoprotein and E2 glycoprotein were the targets for neutralizing antibodies and related to immune protection against CSF. Interleukin-2 (IL2), as an adjuvant, also had been used in CSF vaccine research. In this study, coexpression of the CSFV E0, E2, and IL2 genes by HAdV-5 (rAdV-E0-E2-IL2) was constructed and immunized to evaluate its efficacy. Three expressed genes had been sequentially connected with foot-and-mouth disease virus 2A (FMDV 2A). The vaccine was administered by intramuscular inoculation to CSFV-free pigs (10(8) TCID50) twice at triweekly intervals. No adverse clinical signs were observed in any of the pigs after vaccination. The vaccine induced strong humoral and cellular responses that led to complete protection against clinical signs of lethal CSFV infection, viremia, and shedding of challenge virus. The rAdV-E0-E2-IL2 is a promising, efficient, and safe marker vaccine candidate against CSFV.

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication.

Construction of Foot-and-mouth disease virus 2A-based bicistronic expression vector and coexpression of two genes in goat mammary epithelial cells

Using animal mammary glands as bioreactors for producing commercially important proteins is a cutting-edge direction in the field of biotechnology development and application. Dairy goats are an important dairy livestock, with roughage-resistance, fast propagation, long lactation periods and high milk production per bodyweight; these characteristics make dairy goats ideal for use as mammary gland bioreactors. Foot-and-mouth disease virus 2A (FMDV 2A) is an efficient viral cleavage element that mediates proteolytic cleavage independent of the presence of other FMDV sequences. It is often incorporated into recombinant vectors to generate cleavage in the presence of heterologous sequences. To achieve specific co-expression of two heterologous genes in goat mammary gland epithelial (GMGE) cells, a mammary gland-specific bicistronic expression vector, pFIEβ, containing the β-casein 5′ flanking sequence and FMDV 2A, was successfully constructed and the specific expression of human interleukin 2 (hIL-2) and enhanced green fluorescent protein (EGFP) was conducted in primary GMGE cells. Another bicistronic expression vector, pFIEC, driven by the cytomegalovirus promoter, was constructed as a positive control. In cells transfected with pFIEβ and pFIEC, RT-PCR verified the existence of recombinant fusion mRNA of hIL-2 upstream of EGFP within the FMDV 2A cassette fragment and western blot analysis showed the existence of the fusion between hIL-2 and EGFP. It is concluded that FMDV 2A generated specific co-expression of multiple genes for the first time in primary GMGE cells driven by the β-casein promoter.

Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide

Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly ‘cleaved’ at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (∼100-fold) increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology.

Coordinate lentiviral expression of Cre recombinase and RFP/EGFP mediated by FMDV 2A and analysis of Cre activity

The site-specific recombination mediated by Cre recombinase has been utilized extensively in genetic engineering and gene function studies. Efficient delivery of a Cre enzyme with enzymatic activity and the ability to monitor the enzyme expression are required in applications, and lentiviral constructs with a fluorescent protein (FP) to report the Cre expression are suitable for most studies. However, the current lentiviral vector systems have some deficiencies in precise reporting the Cre expression through fluorescence. To solve the problem, we generated a lentiviral system with Cre and RFP or EGFP bridged by an FMDV 2A sequence in an open reading frame expressed by a CMV promoter. We then examined the capabilities of the constructs to package with VSVG into infectious virus and to mediate expression of the Cre enzyme and fluorescent reporter. Furthermore, we monitored the bioactivities of the expressed products. We demonstrated the coordinate expression of the enzyme and the reporter. The expressed Cre was efficient at removing LoxP-flanked fragments in cells and did not show obvious cellular toxicity, and the expressed FPs allowed direct observation under fluorescent microscope. Therefore, the conjugation of CMV-Cre-2A-FP represents a significant improvement to the current lentiviral Cre delivery systems for obtaining a required Cre activity while accurately monitoring its presence. Our study also provides information concerning application of the established vector system.

Stable expression and purification of a functional processed Fab′ fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor

Monoclonal antibodies and derivative formats such as Fab′ fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab′ fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab′ fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab′ fragment against the Fas receptor, routinely expressed in 1–2 mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab′ fragments.

Generation and characterization of an N pro-disrupted marker bovine viral diarrhea virus derived from a BAC cDNA

In vitro studies showed that N pro protein of bovine viral diarrhea virus (BVDV) interferes with cellular antiviral defense. To understand the role of N pro protein in successful viral invasion of the host and establishment of the lifetime persistence, an infectious N pro-disrupted virus with a noncytopathic (NCP) background is desired. In this study, an N pro-disrupted cDNA, pBSD1-N pro/eGFP2A, was constructed based on an infectious full-length BAC cDNA clone of NCP BVDV strain SD1, pBSD1. In this clone, whole N pro gene except its first 57 nucleotides (nt) was in frame substituted with an eGFP2A sequence. eGFP2A was constructed by in frame fusing a foot-and-mouth disease virus 2A protease (FMDV 2A pro) to C-terminus of eGFP. Intramolecular cleavage of FMDV 2A pro at its C-terminal glycine–proline dipeptide will release the viral nucleocapsid protein from the nascent viral polyprotein and the processed eGFP2A protein will then act as a marker protein. The resulting BAC cDNA clone was propagated stably for at least 10 passages in E. coli strain XL1-blue as determined by sequencing the progeny plasmids. The rescued virus, BSD1-N pro/eGFP2A, showed a peak virus titer approximately 1.2 log 10 lower and a maximum virus yield about 20 hr later than wt SD1, respectively, and was similar to wt SD1 in viral RNA replication and protein expression. FACS, fluorescent microscopy and western blotting assays confirmed that functional eGFP2A protein was expressed and processed properly in MDBK cells. In summary, the availability of BSD1-N pro/eGFP2A with a stable viral genome would facilitate the investigation of the role of N pro protein in transplacental transfer of BVDV and establishment of persistent infection in bovine fetus.

Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris

The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the alcohol oxidase (AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with 0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was 'cleaved' to each functional protein. The yeasts transformed the fused genes, which were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis genes introduced.

Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone.

Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology 2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

462. Comparison of EMCV IRES and FMDV 2A Cleavage Factor for Expression of Canine MGMT and a Second Transgene Using HIV-1 Vectors

We previously described the cloning and expression of canine methylguanine-DNA methyl transferase (MGMT) using monocistronic HIV-1 and gamma-retroviral vectors (Zaboikin M et. al. (2004) Hum Gene Ther 15:383-392). In that study, we showed that the canine MGMT was as efficient as the human MGMT for providing resistance to nitrosourea drugs such as BCNU. Here, we describe the creation and testing of dual-gene expression HIV-1 vectors encoding canine MGMT and EGFP under control of the EF1α promoter.

FMDV–2A sequence and protein arrangement contribute to functionality of CYP2B1–reporter fusion protein

Two optimized forms of green fluorescence proteins (GFP), enhanced GFP (EGFP) and humanized Renilla GFP (hrGFP), were used to track expression of cytochrome P450 2B1 (CYP2B1), an endoplasmic reticulum membrane-bound protein. In transiently expressing HEK293 cells we show that CYP2B1–GFP fusion proteins are stable and functional, whereas the vice-versa-arranged GFP–CYP2B1 fusions are not. The CYP2B1–hrGFP fusion protein is characterized by reduction in mean fluorescence intensity (MFI) to less than 20% of that of the hrGFP protein alone, accompanied by a 50% loss of CYP2B1 activity. Exchanging the linker for an α-helical peptide structure between CYP2B1 and hrGFP does not improve fusion protein activity. Insertion of a short linker (five amino acids) increases reporter protein fluorescence intensity twofold without improving CYP2B1 activity. Introduction of the foot and mouth disease virus 2A sequence providing cotranslational cleavage led to an unstable hrGFP–2A protein, whereas the corresponding EGFP–2A protein was stable and yielded an MFI superior to those of all other fusion constructs tested. CYP2B1 activity of the EGFP–2A–CYP2B1 protein was in the range of that of the unmodified CYP2B1. These data indicate that the protein arrangement EGFP–2A–CYP2B1 is superior to others, since it is most active and visible, which is essential for an effective tracking of the CYP2B1 enzyme.

Generation of Novel, High Titre, Multi-Cistronic Retroviral Vectors Which Simultaneously Express Individual Proteins in Different Subcellular Locations of Hematopoietic Cells.

As gene therapy strategies become ever more sophisticated, there is a need for vectors which efficiently deliver and express more than one gene product to target cells e.g. to enable in vivo selection; to correct multiple genetic loci; to provide a suicide mechanism for safety purposes. Employing alternate codon usage, we have synthesised a novel version of the FMDV 2A self-processing moiety which encodes the same primary amino-acid sequence as the wild type protein (and which possesses equivalent cleavage activity to the wild type protein). By including this synthetic FMDV 2A sequence in retroviral vectors which also contain the wild type sequence, we are able to facilitate the efficient co-translational separation of 3 different proteins encoded by a single transcript. This vector configuration also permits the inclusion of an IRES sequence and thus expression of a fourth protein. Using this technology we have created two tetracistronic retroviral vectors which express eGFP; O6-methylguanine DNA-methyltransferase and truncated human nerve growth factor receptor courtesy of 2A mediated cleavage, and a mitochondrially targeted dsRED via an IRES. Stable amphotropic producer lines containing these vectors generated supernatant which contained a high titre of retroviral particles (>106 IU/ml) which was sufficient for the transduction of K562 human erythroleukemic cells. Analysis of transduced cells confirmed that all four proteins were expressed individually and segragated to their correct subcellular locations. Furthermore, limiting dilution assay revealed that proteins expressed courtesy of the 2A sequences were still generated at high levels, even when cells were infected with a low MOI of virus. We propose that this technology will allow the creation of more sophisticated vectors for use in gene therapy of hematopoietic and other cells.