Bicistronic Expression Vector Research Articles

Alternative routes for production of the drug candidate d-chiro-inositol with Corynebacterium glutamicum using endogenous or promiscuous plant enzymes

d-chiro-Inositol (DCI) is a promising drug candidate for treating insulin resistance and associated diseases such as type 2 diabetes or polycystic ovary syndrome. In this study, we developed two production processes for DCI using Corynebacterium glutamicum as host. In the first process, myo-inositol (MI) is oxidized to 2-keto-myo-inositol (2KMI) by the inositol dehydrogenase (IDH) IolG and then isomerized to 1-keto-d-chiro-inositol (1KDCI) by the isomerases Cg0212 or Cg2312, both of which were identified in this work. 1KDCI is then reduced to DCI by IolG. Overproduction of IolG and Cg0212 in a chassis strain unable to degrade inositols allowed the production of 1.1 g/L DCI from 10 g/L MI. As both reactions involved are reversible, only a partial conversion of MI to DCI can be achieved. To enable higher conversion ratios, a novel route towards DCI was established by utilizing the promiscuous activity of two plant-derived enzymes, the NAD+-dependent d-ononitol dehydrogenase MtOEPa and the NADPH-dependent d-pinitol dehydrogenase MtOEPb from Medicago truncatula (barrelclover). Heterologous production of these enzymes in the chassis strain led to the production of 1.6 g/L DCI from 10 g/L MI. For replacing the substrate MI by glucose, the two plant genes were co-expressed with the endogenous myo-inositol-1-phosphate synthase gene ino1 either as a synthetic operon or using a novel, bicistronic T7-based expression vector. With the single operon construct, 0.75 g/L DCI was formed from 20 g/L glucose, whereas with the bicistronic construct 1.2 g/L DCI was obtained, disclosing C. glutamicum as an attractive host for of d-chiro-inositol production.

Promoting soluble expression of hybrid mussel foot proteins by SUMO-TrxA tags for production of mussel glue

Mussel foot proteins (Mfps) display application potential with strong adhesion, enabling mussels to adhere firmly to various surfaces. Mytilus galloprovincialis foot protein 3B (Mgfp-3B) exhibits this characteristic remarkably. However, it remains a challenge for further research due to the low soluble expression of heterologous production. In this study, a small ubiquitin-related modifier (SUMO) and thioredoxin A (TrxA), which catalyzed the proper folding of disulfide bridges, were selected to increase the soluble expression of mfps. An additional ribosome binding site was introduced between the molecular chaperones and Mgfp-3B (fp-3) to form a bicistronic translation-coupled expression vector for co-expression. The results revealed that the combination of SUMO-TrxA increased the soluble expression of fp-3 by 18.07 %. Furthermore, the SUMO-TrxA also boosted the soluble expression of hybrid mfps Mgfp-3B-Mfp-1 (fp-3-1) by 11.29 %, Mgfp-3B-Mgfp-3B (fp-3-3) by 19.91 %, and Mgfp-3B-Mgfp-5 (fp-3-5) by 14.03 %. Ultimately, by high cell density cultivation in a 5 L bioreactor, the yields of fp-3, fp-3-3, and fp-3-5 co-expressed with SUMO-TrxA reached 217.75 mg/L, 127.2 mg/L, and 97.28 mg/L, respectively. Consequently, soluble production of mfps holds great potential for the sustainable supply of protein adhesive materials.

Identification of an IRES within the coding region of the structural protein of human rhinovirus 16.

As an alternative mechanism for cap-dependent (m7GpppN) translation, internal ribosome entry site (IRES)-dependent translation has been observed in the 5' untranslated regions (5' UTR) and coding regions of a number of viral and eukaryotic mRNAs. In this study, a series of 5' terminal truncated structural protein genes that were fused with GFP was used to screen for potential IRESs, and IRESs were identified using a bicistronic luciferase vector or GFP expression vector possessing a hairpin structure. Our results revealed that a putative IRES was located between nt 1982 and 2281 in the VP3 coding region of the human rhinovirus 16 (HRV16) genomes. We also demonstrated that effective IRES-initiated protein expression in vitro did not occur through splicing sites or cryptic promoters. We confirmed that thapsigargin (TG), an inducer of endoplasmic reticulum stress (ERS), facilitated increased IRES activity in a dose-dependent manner. Additionally, the secondary structure of the IRES was predicted online using the RNAfold web server.

Induction of biosynthesis of ketocarotenoid from β-carotene in fish embryos

Ketocarotenoids are high-value molecules with powerful antioxidant properties. They are widely used as a feed supplements due to their vital functions for animals, particularly for fish and marine animals. However, fish is not able to synthesize ketocarotenoids but consume them from diet. Here, we report metabolic engineering of fish cells and fish embryos for biosynthesis of ketocarotenoid from β-carotene as a substrate. Firstly, we introduced two sets of fish-codon optimized genes (crtW and crtZ) from Brevundimonas sp., and (crtS and crtR) from Xanthophyllomyces dendrorhous, to the cultured common carp cells (Epithelioma papulosum cyprinid, EPC cells) to test the production of ketocarotenoids in vitro. We then introduced the optimized set of genes, crtW and crtZ, to zebrafish (Danio rerio) embryos and found that they could efficiently mediate the synthesis of ketocarotenoids from β-carotene in vivo. Subsequently, we constructed a bicistronic expression vector containing crtW and crtZ genes linked with a P2A peptide coding sequence, and evaluated the production of ketocarotenoids using both in vitro and in vivo systems. Overall, we unprecedentedly stimulated fish cells and zebrafish embryos to synthesize ketocarotenoids with a total content of 29.9 μg/g DW and 16.46 μg/g DW, respectively. Furthermore, we successfully optimized the ketocarotenoids biosynthesis and significantly increased the total ketocarotenoids content in fish models up to 2.5 and 2.14 fold, corresponding to 75.24 and 35.25 μg/g DW, respectively. This study is a proof of concept of engineering animals to manufacture ketocarotenoids and provides a pilot study to enable fish to synthesize ketocarotenoids in vivo.

Pyruvate Kinase M2 Coordinates Metabolism Switch between Glycolysis and Glutaminolysis in Cancer Cells.

SummaryCancer cells alter their nutrition metabolism to cope the stressful environment. One important metabolism adjustment is that cancer cells activate glutaminolysis in response to the reduced carbon from glucose entering into the TCA cycle due to inactivation of several enzymes in glycolysis. An important question is how the cancer cells coordinate the changes of glycolysis and glutaminolysis. In this report, we demonstrate that the pyruvate kinase inactive dimer PKM2 facilitates activation of glutaminolysis. Our experiments show that growth stimulations promote PKM2 dimer. The dimer PKM2 plays a role in regulation of glutaminolysis by upregulation of mitochondrial glutaminase I (GLS-1). PKM2 dimer regulates the GLS-1 expression by controlling internal ribosome entry site (IRES)-dependent c-myc translation. Growth stimulations promote PKM2 interacting with c-myc IRES-RNA, thus facilitating c-myc IRES-dependent translation. Our study reveals an important linker that coordinates the metabolism adjustment in cancer cells.

Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system

BackgroundCorynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum.ResultsIn this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L.ConclusionIn this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries

BackgroundAntibody fragments can be expressed in Escherichia coli, where they are commonly directed to the periplasm via Sec pathway to enable disulphide bridge formations and correct folding. In order to transport antibody fragments to the periplasmic space via Sec pathway, they are equipped with N-terminal signal sequence. Periplasmic expression has many benefits but it’s also subjected to many hurdles like inefficient translocation across the inner membrane and insufficient capacity of the translocation system. One solution to overcome these hurdles is a modulation of codon usage of signal sequence which has proved to be an efficient way of tuning the translocation process. Modulation of codon usage of signal sequences has been successfully employed also in improving the expression levels of antibody fragments, but unfortunately the effect of codon usage on the expression has not been thoroughly analyzed.ResultsIn the present study we established three synonymous PelB signal sequence libraries by modulating codon usage of light chain and heavy chain PelB signal sequences of a Fab fragment. Each region (n-region, hydrophobic region and c-region) of the PelB signal sequence in the both chains of the Fab fragment in a bicistronic expression vector was mutated separately. We then screened for clones with improved expression profile. The best source for improved clones was the n-region library but in general, improved clones were obtained from all of the three libraries. After screening, we analyzed the effects of codon usage and mRNA secondary structures of chosen clones on the expression levels of the Fab fragment. When it comes to codon usage based factors, it was discovered that especially codon usage of fifth leucine position of the light chain PelB affects the expression levels of Fab fragment. In addition, we observed that mRNA secondary structures in the translation initiation regions of the light and heavy chain have an effect on expression levels as well.ConclusionsIn conclusion, the established synonymous signal sequence libraries are good sources for discovering Fab fragments with improved expression profile and obtaining new codon usage related information.

Production of breast cancer cell lines expressing viral gene VIF

Introduction: Virion Infectivity factor (Vif) is a protein required for lentiviruses successful infection of human target cells, through the degradation of host restriction factors, APOBEC3 [1]. In the last years, the activity of APOBEC3 deaminases has also been associated with mutation patterns found in cancer cells, including breast cancer cells [2]. We hypothesize that Vif protein can be used to inhibit APOBEC3 avoiding breast cancer progression and resistance to chemotherapy. This hypothesis should be tested in breast cancer cells expressing Vif proteins from HIV-1 (Vif1), and HIV-2 (Vif2) treated with chemotherapy agents. The aim of this work was the production of breast cell lines expressing constitutively Vif1 and Vif 2 proteins, using lentiviruses as a gene-delivering system. Materials and methods: Vif1 and Vif2 delivering lentivirus vectors were obtained using the fourth generation lentiviral packaging systems (Clontech). Briefly, Vif 1 and Vif 2 genes were cloned in a bicistronic lentiviral expression vector (pLVX-IRES-zsGreen1) which allowed the co-expression of Vif and the reporter gene (ZsGreen) from a single mRNA transcript. VSV-G pseudotyped lentiviruses were produced by transfection of packaging cells, Lenti-X 293T cells (Clontech). Viruses were then used to transduce MCF10A and HCC1806 breast cells. Transduced cells stably expressing fluorescent marker were isolated by Fluorescence Activated Cell Sorting (FACS-AriaIII). The isolated cells were expanded and characterized. RNA was extracted from the cell lines MCF10A-Vif and HCC1806-Vif, and cDNA synthesis was performed. Expression of Vif was confirmed by PCR amplification of cDNA and APOBEC3G, 3B and 3C expression levels were also confirmed by qRT-PCR (TaqMan Technology). Results: We obtained high-titer of Vif delivering lentiviruses used to transduce the breast cells. FACS allowed us to obtain sorting populations with highly purified transduced cells (sorting populations with 97 to 99% purity). Characterization of the new cell populations showed that lentiviral constructs integrated either Vif1 or Vif2 genes into genomic DNA. Moreover, Vif genes are expressed in these populations with no significant effect on the mRNA levels of APOBEC3G, 3C, and 3B (∼1.5 to 2 fold increases). Discussion and conclusions: The genomic DNA of MCF10A (normal breast cell line) and HCC1806 (breast cancer cell line) were modified with Vif gene (and ZsGreen gene) integration generating new stable breast cell lines. In the near future, we will be able to confirm or not that inhibition of APOBEC3 by Vif sensitize to chemotherapy the breast cancer cells, and hopefully decrease recurrent relapses. We believe that the use of Vif to inhibit APOBEC3 is a promising therapeutic tool model with application in breast cancer disease.

Co-expression of IL-15 enhances anti-neuroblastoma effectivity of a tyrosine hydroxylase-directed DNA vaccination in mice

Long-term survival of high-risk neuroblastoma (NB) patients still remains under 50%. Here, we report the generation, in vitro characterization and anti-tumor effectivity of a new bicistronic xenogenic DNA vaccine encoding tyrosine hydroxylase (TH) that is highly expressed in NB tumors, and the immune stimulating cytokine interleukin 15 (IL-15) that induces cytotoxic but not regulatory T cells. The DNA sequences of TH linked to ubiquitin and of IL-15 were integrated into the bicistronic expression vector pIRES. Successful production and bioactivity of the vaccine-derived IL-15- and TH protein were shown by ELISA, bioactivity assay and western blot analysis. Further, DNA vaccine-driven gene transfer to the antigen presenting cells of Peyer’s patches using attenuated Salmonella typhimurium that served as oral delivery system was shown by immunofluorescence analysis. The anti-tumor effect of the generated vaccine was evaluated in a syngeneic mouse model (A/J mice, n = 12) after immunization with S. typhimurium (3× prior and 3× after tumor implantation). Importantly, TH-/IL-15-based DNA vaccination resulted in an enhanced tumor remission in 45.5% of mice compared to controls (TH (16.7%), IL-15 (0%)) and reduced spontaneous metastasis (30.0%) compared to controls (TH (63.6%), IL-15 (70.0%)). Interestingly, similar levels of tumor infiltrating CD8+ T cells were observed among all experimental groups. Finally, co-expression of IL-15 did not result in elevated regulatory T cell levels in tumor environment measured by flow cytometry. In conclusion, co-expression of the stimulatory cytokine IL-15 enhanced the NB-specific anti-tumor effectivity of a TH-directed vaccination in mice and may provide a novel immunological approach for NB patients.

A secretary bi-cistronic baculovirus expression system with improved production of the HA1 protein of H6 influenza virus in insect cells and Spodoptera litura larvae

A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype.

Impairment of a membrane-targeting protein translated from a downstream gene of a “self-cleaving” T2A peptide conjunction

The requirement for reliable bicistronic or multicistronic vectors in gene delivery systems is at the forefront of bio/biomedical technology. A method that provides an efficient co-expression of multiple heterologous proteins would be valuable for many applications, especially in medical science for treating various types of disease. In this study, we designed and constructed a bicistronic expression vector using a self-cleaving 2A peptide derived from a virus of the insect Thosea asigna (T2A). This exhibited the most efficient cleavage of the 2A sequence. Two versions of the T2A-based vector were constructed by switching the DNA sequences encoding the proteins of interest, the N-myristoylated protein and the nuclear-homing protein, upstream and downstream of the 2A linker, respectively. Our results showed that similar levels of mRNA expression were found and 100% of cleavage efficiency of T2A was observed. Nevertheless, we also reported the cleared evidence that the N-myristoylated protein cannot be placed downstream of the 2A sequence. Since the protein product fails to translocate to the plasma membrane due to altered myristoylation process, the gene position of the T2A-based vector is meaningful for the subcellular localization of the N-myristoylated protein. Therefore, the observation was marked as a precaution for using the 2A peptide. To adopt the 2A peptide technology for generating the bicistronic or multicistronic expression, the vector design should be carefully considered for the transgene position, signal sequences, and post-translational modifications of each individual protein.

The effects of nucleotide usage in key nucleotide positions +4 and -3 flanking start codon on translation levels mediated by IRES of hepatitis C virus.

Internal ribosomal entry site (IRES) functions as a cis-acting RNA element, which drives an alternative and cap-independent translation initiation pathway. Currently, there are few studies on effects of nucleotide usages at key nucleotide positions +4 and -3 flanking start codon mediated by IRES of hepatitis C virus (HCV). Herein, we focus on the effect of nucleotide usages at -3 and +4 positions mediated by HCV IRES. The nucleotide contexts flanking AUG start codon employed by HCV IRES is firstly analyzed. We found that each position in the six nucleotide positions (-4 to +6) flanking start codon of HCV has a strong tendency to select the specific nucleotide. A set of bicistronic expression vectors containing CAT gene, HCV IRES and EGFP gene were constructed, including 16 different nucleotide combinations at position -3 and +4. Each set, in which nucleotide at the -3 and +4 position has been changed into different nucleotides, included 16 types of bicistronic expression vectors. It was found that the purine nucleotide at the position -3 or +4 obviously impacts on HCV IRES-related expression, and IRES-driven translation is potentially influenced by the Kozak rule. Our results suggest that optimization of nucleotides at positions -3 and +4 is a convenient and efficient way to enhance the level of IRES-mediated translation. Keywords: Cap-independent translation; internal ribosomal entry site; hepatitis C virus; bicistronic expression vector; translation efficiency.

Abstract IA29: Targeting undruggable proteins for degradation with small molecules

Abstract Many well-studied oncoproteins are currently viewed to be “undruggable”. The infamous teratogen thalidomide was serendipitously discovered to be a potent antimyeloma drug in the 1990s but its mechanism of action was unknown. In 2010 thalidomide was shown by Handa and coworkers to bind to cereblon, which is the substrate-recognition subunit of a cullin-dependent ubiquitin ligase. Moreover, thalidomide was found to inhibit this ligase in vitro, suggesting that thalidomide is a cereblon antagonist. However, it soon became apparent that loss of cereblon rendered myeloma cells resistant to thalidomide (and related drugs such as lenalidomide, collectively referred to as IMiDs). This suggested to us that cereblon, once bound to an IMiD, acquires a new (neomorphic) ability to target a myeloma-relevant protein for destruction. To look for such a protein we built a bicistronic expression vector that encodes both a protein open reading frame (ORF)-firefly luciferase fusion protein and, from the same mRNA, renilla luciferase for normalization purposes, with both proteins under the control of an internal ribosomal entry site (IRES). We then made a library of ~12,000 such ORF-luciferase fusions and introduced them into 293T cells in quadruplicate in multiwell plate format (1 ORF/well), treated the cells with lenalidomide or vehicle, and monitored changes in firefly luciferase/renilla luciferase as a surrogate for changes in ORF stability. In this way we discovered that cereblon, once bound to lenalidomide, destabilizes IKZF1 and IKZF3. We went on to show that downregulation of IKZF1 and IKZF3 is intimately linked to the antimyeloma activity of the IMiDs. Ben Ebert and his group working in parallel and using complimentary approaches reached the same conclusion. Published and unpublished work from investigators such as Jay Bradner and Craig Crews suggests IMiDs can be reengineered to serve as molecular matchmakers between cereblon and novel targets. We are currently developing new screening methodologies to identify chemicals and genetic perturbants that directly or indirectly destabilize proteins of interest. Citation Format: William G. Kaelin, Jr. Targeting undruggable proteins for degradation with small molecules. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA29.

Selective and augmented β-glucuronidase expression combined with DOX-GA3 application elicits the potent suppression of prostate cancer.

The present study was carried out to evaluate the specific and amplified β-glucuronidase (βG) expression in prostate cancer cells by using a prostate‑specific antigen (PSA) promoter-controlled bicistronic adenovirus and to evaluate the specific killing of prostate cancer cells after the application of the prodrug DOX‑GA3. Bicistronic adenoviral expression vectors were constructed, and the effectiveness of specific and amplified expression was evaluated using luciferase and EGFP as reporter genes. βG expression was detected in LNCaP cells after they were infected with the βG‑expressing PSA promoter-controlled bicistronic adenovirus. MTT assays were conducted to evaluate the cytoxicity on the infected cells after the application of the prodrug DOX‑GA3. Tumor growth inhibition was also evaluated in nude mice after treatment with the βG‑expressing adenovirus and DOX‑GA3. Selective and amplified expression was observed in the PSA-producing LNCaP cells, but not in the PSA‑non‑producing DU145 cells. Potent cytotoxity and a strong bystander effect were observed in the LNCaP cells after infection with the βG‑expressing adenovirus and the application of DOX‑GA3. Intravenous injection of a GAL4 regulated bicistronic adenovirus vector constructed to express βG under the control of the PSA promoter (Ad/PSAP‑GV16‑βG) and the application of DOX‑GA3 strongly inhibited tumor growth and prolonged the survival time of tumor‑bearing nude mice. Selective and amplified βG expression together with the prodrug DOX‑GA3 had an increased antitumor effect, showing great potential for prostate cancer therapy.

Identification of Rhopalosiphum Padi Virus 5' Untranslated Region Sequences Required for Cryptic Promoter Activity and Internal Ribosome Entry.

The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector.

HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data.

Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.

In-vitro Characterization and Evaluation of Apoptotic Potential of Bicistronic Plasmid Encoding HN Gene of Newcastle Disease Virus and Human TNF-α

The viral gene oncotherapy in combination with cytokines emerges as an exciting strategy for cancer therapy due to its minimal side effects and tumor specificity. HN is the surface protein of NDV which is involved in virus infectivity and is known to kill many cancerous cell types. TNF-α, a multifactorial cytokine has direct anti-tumor activity by activating the extrinsic pathways of apoptosis. In the present study, HN gene of NDV and TNF-α of human were cloned at multiple cloning sites (MCS) 1 and 2 of bicistronic expression vector pVIVO2. Expression pattern of recombinant clone was checked on transcriptional and translational level by RT-PCR, Immunofluorescence assay and flow cytometry. On flow cytometric analysis HN gene expression was found to be 28.30 ± 1.21; 5.22 ± 0.60%, and TNF-α gene expression was found to be 15.44 ± 0.42; 6.51 ± 0.757%, in HeLa cells transfected with pVIVO.nd.hn.hu.tnf and pVIVO2 empty vector control, respectively. These assays confirm that HN and TNF-α act synergistically in the induction of apoptosis in HeLa cells.

S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation.

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5′-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients.

Low False-Positives in an mLumin-Based Bimolecular Fluorescence Complementation System with a Bicistronic Expression Vector

The simplicity and sensitivity of the bimolecular fluorescence complementation (BiFC) assay make it a powerful tool to investigate protein-protein interactions (PPIs) in living cells. However, non-specific association of the fluorescent protein fragments in a BiFC system can complicate evaluation of PPIs. Here, we introduced a bicistronic expression vector, pBudCE4.1, into an mLumin-based BiFC system, denoted as the BEVL-BiFC system. The BEVL-BiFC system achieved a 25-fold contrast in BiFC efficiency between positive (Fos/Jun) and negative (ΔFos/Jun) PPIs. The high BiFC efficiency was due to a low false-positive rate, where less than 2% of cells displayed BiFC in the negative control. K-Ras and its interactive proteins, Ras binding domain (RBD) of Raf-1 and Grb2 were used to confirm the accuracy of the BEVL-BiFC system. The results also provide direct evidence in individual cells that post-translational modification of K-Ras and its localization at the plasma membrane (PM) were not essential for the interaction of K-Ras and Raf-1, whereas the interaction of Grb2 and K-Ras did depend on the PM localization of K-Ras. Taken together, the BEVL-BiFC system was developed to reduce the false-positive phenomenon in BiFC assays, resulting in more robust and accurate measurement of PPIs in living cells.