Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast.

CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.

Synthesis of α-Haloboronates by the Halogenation of gem-Diborylalkanes via Tetracoordinate Boron Species.

α-Haloboronates have a wide range of applications in organic chemistry as synthetic synthons; however, traditional synthetic methods of α-haloboronates are harsh and complicated. Herein, we used nBuLi as the nucleophilic reagent to attack the boron atom in gem-diborylalkanes to form tetracoordinate boron species and successfully achieved α-chloroboronates and α-bromoboronates with readily accessible electrophilic halogen reagents (NCS and NBS). The reaction is transition-metal-free and features a broad substrate scope and diversified valuable products.

The Study of Overexpression of Peroxiredoxin-2 Reduces MPP+-Induced Toxicity in the Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by dopaminergic neuron loss, which is related to excessive reactive oxygen species (ROS) accumulation. Endogenous peroxiredoxin-2 (Prdx-2) has potent anti-oxidative and anti-apoptotic effects. Proteomics studies revealed plasma levels of Prdx-2 were significantly lower in PD patients than in healthy individuals. For further study of the activation of Prdx-2 and its role in vitro, SH-SY5Y cells and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) were used to model PD. ROS content, mitochondrial membrane potential, and cell viability were used to assess the effect of MPP+ in SH-SY5Y cells. JC-1 staining was used to determine mitochondrial membrane potential. ROS content was detected using a DCFH-DA kit. Cell viability was measured using the Cell Counting Kit-8 assay. Western blot detected the protein levels of tyrosine hydroxylase (TH), Prdx-2, silent information regulator of transcription 1 (SIRT1), Bax, and Bcl-2. The results showed that MPP+-induced accumulation of ROS, depolarization of mitochondrial membrane potential, and reduction of cell viability occurred in SH-SY5Y cells. In addition, the levels of TH, Prdx-2, and SIRT1 decreased, while the ratios of Bax and Bcl-2 increased. Then, Prdx-2 overexpression in SH-SY5Y cells showed significant protection against MPP+ -induced neuronal toxicity, as evidenced by the decrease in ROS content, increase in cell viability, increase in the level of TH, and decrease in the ratios of Bax and Bcl-2. Meanwhile, SIRT1 levels increase with the level of Prdx-2. This suggests that the protection of Prdx-2 may be related to SIRT1. In conclusion, this study indicated that overexpression of Prdx-2 reduces MPP+-induced toxicity in SH-SY5Y cells and may be mediated by SIRT1.

Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

Fast arsenate As(V) adsorption and removal from water using aluminium Al(III) fixed on Kapok fibres.

Arsenic (As) is among the most dangerous metalloids and is harmful to human wellbeing. In this laboratory study, Al(III)-modified kapok fibres (Al-Kapok) were used to remove As(V) from water. The sorbent was characterised using Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Batch experiments were performed to observe the performance of Al-Kapok in the removal of As(V) and to examine the effects of pH, temperature, adsorbent dose, and coexisting ions on the adsorption process. The surface of the sorbent changed after aluminium modification, and the results of the batch experiments showed that the adsorption of As(V) occurred mainly via endothermic-spontaneous chemisorption at the solution and solid interface of Al-Kapok. The As(V) removal efficiency was approximately 76%-84%, and it was slightly affected at pH levels below 8.0. Further study showed that the maximum adsorption capacity of Al-Kapok for As(V) was 118 µg/g at 30 °C and pH 6, and notable adverse effects were caused by the presence of SO42-and PO43-. It was also found that the boundary layer and film diffusion contributed more to As(V) adsorption. After five adsorption/desorption cycles, regeneration recovered approximately 92% of the adsorption capacity of Al-Kapok used. Overall, Al-Kapok appears to be a suitable adsorbent material for the purification of As-contaminated water.

Photo-induced trifunctionalization of bromostyrenes via remote radical migration reactions of tetracoordinate boron species.

Tetracoordinate boron species have emerged as radical precursors via deboronation by photo-induced single electron transfer (SET) pathway. These reactions usually produce an alkyl radical and boron-bound species, and the valuable boron species are always discarded as a by-product. Given the importance of boron species, it will be very attractive if the two parts could be incorporated into the eventual products. Herein we report a photo-catalyzed strategy in which in situ generated tetracoordinated boron species decomposed into both alkyl radicals and boron species under visible light irradiation, due to the pre-installation of a vinyl group on the aromatic ring, the newly generated alkyl radical attacks the vinyl group while leaving the boron species on ipso-position, then both radical part and boron moiety are safely incorporated into the final product. Tertiary borons, secondary borons, gem-diborons as well as 1,2-diborons, and versatile electrophiles are all well tolerated under this transformation, of note, ortho-, meta- and para-bromostyrenes all demonstrated good capabilities. The reaction portraits high atom economy, broad substrate scope, and diversified valuable products with tertiary or quaternary carbon center generated, with diborons as substrates, Csp2-B and Csp3-B are established simultaneously, which are precious synthetic building blocks in chemical synthesis.

Structural features determining the vitamin K epoxide reduction activity in the VKOR family of membrane oxidoreductases.

Vitamin K epoxide reductases (VKORs) are a large family of integral membrane enzymes found from bacteria to humans. Human VKOR, specific target of warfarin, has both the epoxide and quinone reductase activity to maintain the vitamin K cycle. Bacterial VKOR homologs, however, are insensitive to warfarin inhibition and are quinone reductases incapable of epoxide reduction. What affords the epoxide reductase activity in human VKOR remains unknown. Here, we show that a representative bacterial VKOR homolog can be converted to an epoxide reductase that is also inhibitable by warfarin. To generate this new activity, we first substituted several regions surrounding the active site of bacterial VKOR by those from human VKOR based on comparison of their crystal structures. Subsequent systematic substitutions narrowed down to merely eight residues, with the addition of a membrane anchor domain, that are responsible for the epoxide reductase activity. Substitutions corresponding to N80 and Y139 in human VKOR provide strong hydrogen bonding interactions to facilitate the epoxide reduction. The rest of six substitutions increase the size and change the shape of the substrate-binding pocket, and the membrane anchor domain stabilizes this pocket while allowing certain flexibility for optimal binding of the epoxide substrate. Overall, our study reveals the structural features of the epoxide reductase activity carried out by a subset of VKOR family in the membrane environment.

Passerini-type reaction of boronic acids enables α-hydroxyketones synthesis.

Multicomponent reactions (MCRs) facilitate the rapid and diverse construction of molecular scaffolds with modularity and step economy. In this work, engagement of boronic acids as carbon nucleophiles culminates in a Passerini-type three-component coupling reaction towards the synthesis of an expanded inventory of α-hydroxyketones with skeletal diversity. In addition to the appealing features of MCRs, this protocol portrays good functional group tolerance, broad substrate scope under mild conditions and operational simplicity. The utility of this chemistry is further demonstrated by amenable modifications of bioactive products and pharmaceuticals as well as in the functionalization of products to useful compounds.

Characterization of missense mutations in the signal peptide and propeptide of FIX in hemophilia B by a cell-based assay.

Many mutations in the signal peptide and propeptide of factor IX (FIX) cause hemophilia B. A FIX variants database reports 28 unique missense mutations in these regions that lead to FIX deficiency, but the underlying mechanism is known only for the mutations on R43 that interfere with propeptide cleavage. It remains unclear how other mutations result in FIX deficiency and why patients carrying the same mutation have different bleeding tendencies. Here, we modify a cell-based reporter assay to characterize the missense mutations in the signal peptide and propeptide of FIX. The results show that the level of secreted conformation-specific reporter (SCSR), which has a functional γ-carboxyglutamate (Gla) domain of FIX, decreases significantly in most mutations. The decreased SCSR level is consistent with FIX deficiency in hemophilia B patients. Moreover, we find that the decrease in the SCSR level is caused by several distinct mechanisms, including interfering with cotranslational translocation into the endoplasmic reticulum, protein secretion, γ-carboxylation of the Gla domain, and cleavage of the signal peptide or propeptide. Importantly, our results also show that the SCSR levels of most signal peptide and propeptide mutations increase with vitamin K concentration, suggesting that the heterogeneity of bleeding tendencies may be related to vitamin K levels in the body. Thus, oral administration of vitamin K may alleviate the severity of bleeding tendencies in patients with missense mutations in the FIX signal peptide and propeptide regions.

Predisposition to Alzheimer's and Age-Related Brain Pathologies by PM2.5 Exposure: Perspective on the Roles of Oxidative Stress and TRPM2 Channel.

Accumulating epidemiological evidence supports that chronic exposure to ambient fine particular matters of <2.5 µm (PM2.5) predisposes both children and adults to Alzheimer's disease (AD) and age-related brain damage leading to dementia. There is also experimental evidence to show that PM2.5 exposure results in early onset of AD-related pathologies in transgenic AD mice and development of AD-related and age-related brain pathologies in healthy rodents. Studies have also documented that PM2.5 exposure causes AD-linked molecular and cellular alterations, such as mitochondrial dysfunction, synaptic deficits, impaired neurite growth, neuronal cell death, glial cell activation, neuroinflammation, and neurovascular dysfunction, in addition to elevated levels of amyloid ß (Aß) and tau phosphorylation. Oxidative stress and the oxidative stress-sensitive TRPM2 channel play important roles in mediating multiple molecular and cellular alterations that underpin AD-related cognitive dysfunction. Documented evidence suggests critical engagement of oxidative stress and TRPM2 channel activation in various PM2.5-induced cellular effects. Here we discuss recent studies that favor causative relationships of PM2.5 exposure to increased AD prevalence and AD- and age-related pathologies, and raise the perspective on the roles of oxidative stress and the TRPM2 channel in mediating PM2.5-induced predisposition to AD and age-related brain damage.

Organization of the Addax Major Histocompatibility Complex Provides Insights Into Ruminant Evolution.

Ruminants are critical as prey in transferring solar energy fixed by plants into carnivorous species, yet the genetic signature of the driving forces leading to the evolutionary success of the huge number of ruminant species remains largely unknown. Here we report a complete DNA map of the major histocompatibility complex (MHC) of the addax (Addax nasomaculatus) genome by sequencing a total of 47 overlapping BAC clones previously mapped to cover the MHC region. The addax MHC is composed of 3,224,151 nucleotides, harboring a total of 150 coding genes, 50 tRNA genes, and 14 non-coding RNA genes. The organization of addax MHC was found to be highly conserved to those of sheep and cattle, highlighted by a large piece of chromosome inversion that divided the MHC class II into IIa and IIb subregions. It is now highly possible that all of the ruminant species in the family of Bovidae carry the same chromosome inversion in the MHC region, inherited from a common ancestor of ruminants. Phylogenetic analysis indicated that DY, a ruminant-specific gene located at the boundary of the inversion and highly expressed in dendritic cells, was possibly evolved from DQ, with an estimated divergence time ~140 million years ago. Homology modeling showed that the overall predicted structure of addax DY was similar to that of HLA-DQ2. However, the pocket properties of P1, P4, P6, and P9, which were critical for antigen binding in the addax DY, showed certain distinctive features. Structural analysis suggested that the populations of peptide antigens presented by addax DY and HLA-DQ2 were quite diverse, which in theory could serve to promote microbial regulation in the rumen by ruminant species, contributing to enhanced grass utilization ability. In summary, the results of our study helped to enhance our understanding of the MHC evolution and provided additional supportive evidence to our previous hypothesis that an ancient chromosome inversion in the MHC region of the last common ancestor of ruminants may have contributed to the evolutionary success of current ruminants on our planet.

TRPM2 channel: A novel target for alleviating ischaemia-reperfusion, chronic cerebral hypo-perfusion and neonatal hypoxic-ischaemic brain damage.

The transient receptor potential melastatin-related 2 (TRPM2) channel, a reactive oxygen species (ROS)-sensitive cation channel, has been well recognized for being an important and common mechanism that confers the susceptibility to ROS-induced cell death. An elevated level of ROS is a salient feature of ischaemia-reperfusion, chronic cerebral hypo-perfusion and neonatal hypoxia-ischaemia. The TRPM2 channel is expressed in hippocampus, cortex and striatum, the brain regions that are critical for cognitive functions. In this review, we examine the recent studies that combine pharmacological and/or genetic interventions with using in vitro and in vivo models to demonstrate a crucial role of the TRPM2 channel in brain damage by ischaemia-reperfusion, chronic cerebral hypo-perfusion and neonatal hypoxic-ischaemia. We also discuss the current understanding of the underlying TRPM2-dependent cellular and molecular mechanisms. These new findings lead to the hypothesis of targeting the TRPM2 channel as a potential novel therapeutic strategy to alleviate brain damage and cognitive dysfunction caused by these conditions.

A high-density BAC physical map covering the entire MHC region of addax antelope genome.

BACKGROUND: The mammalian major histocompatibility complex (MHC) harbours clusters of genes associated with the immunological defence of animals against infectious pathogens. At present, no complete MHC physical map is available for any of the wild ruminant species in the world. RESULTS: The high-density physical map is composed of two contigs of 47 overlapping bacterial artificial chromosome (BAC) clones, with an average of 115 Kb for each BAC, covering the entire addax MHC genome. The first contig has 40 overlapping BAC clones covering an approximately 2.9 Mb region of MHC class I, class III, and class IIa, and the second contig has 7 BAC clones covering an approximately 500 Kb genomic region that harbours MHC class IIb. The relative position of each BAC corresponding to the MHC sequence was determined by comparative mapping using PCR screening of the BAC library of 192,000 clones, and the order of BACs was determined by DNA fingerprinting. The overlaps of neighboring BACs were cross-verified by both BAC-end sequencing and co-amplification of identical PCR fragments within the overlapped region, with their identities further confirmed by DNA sequencing. CONCLUSIONS: We report here the successful construction of a high-quality physical map for the addax MHC region using BACs and comparative mapping. The addax MHC physical map we constructed showed one gap of approximately 18 Mb formed by an ancient autosomal inversion that divided the MHC class II into IIa and IIb. The autosomal inversion provides compelling evidence that the MHC organizations in all of the ruminant species are relatively conserved.

Human Papillomavirus 16 oncoprotein E7 retards mitotic progression by blocking Mps1-MAP4 signaling cascade.

Human epithelial cells can be infected by more than 200 types of human papilloma viruses (HPVs), and persistent HPV infections lead to cervical cancer or other deadly cancers. It has been established that mitotic progression is critical for HPV16 infection, but the underlying mechanism remains unknown. Here, we report that oncoprotein E7 of HPV16 but not HPV18 retards mitotic progression in host cell by direct binding to the C terminus of Microtubule-Associated Protein 4 (MAP4). MAP4 is a novel bona fide target of HPV16E7 protein which binds and recruits the latter to spindle microtubule in mitosis. HPV16E7 protein promotes MAP4 stability by inhibiting MAP4 phosphorylation- mediated degradation to increase the stability of microtubule polymerization and cause an extend mitotic progression. We further uncovered that Mps1 is a kinase of MAP4, and E7-MAP4 binding blocks Mps1 phosphorylation of MAP4, thereby interrupting phosphorylation-dependent MAP4 degradation. Mutations of MAP4 at T927ES928E partially abolished E7-binding capacity and rescued mitotic progression in host cells. In conclusion, our study reveals a molecular mechanism by which HPV16E7 perturbs host mitotic progression by interfering Mps1-MAP4 signaling cascade, which results in an extended infection window and may facilitate the persistent HPV16 infection.

Increasing the TRPM2 Channel Expression in Human Neuroblastoma SH-SY5Y Cells Augments the Susceptibility to ROS-Induced Cell Death.

Human neuroblastoma SH-SY5Y cells are a widely-used human neuronal cell model in the study of neurodegeneration. A recent study shows that, 1-methyl-4-phenylpyridine ion (MPP), which selectively causes dopaminergic neuronal death leading to Parkinson's disease-like symptoms, can reduce SH-SY5Y cell viability by inducing H2O2 generation and subsequent TRPM2 channel activation. MPP-induced cell death is enhanced by increasing the TRPM2 expression. By contrast, increasing the TRPM2 expression has also been reported to support SH-SY5Y cell survival after exposure to H2O2, leading to the suggestion of a protective role for the TRPM2 channel. To clarify the role of reactive oxygen species (ROS)-induced TRPM2 channel activation in SH-SY5Y cells, we generated a stable SH-SY5Y cell line overexpressing the human TRPM2 channel and examined cell death and cell viability after exposure to H2O2 in the wild-type and TRPM2-overexpressing SH-SY5Y cells. Exposure to H2O2 resulted in concentration-dependent cell death and reduction in cell viability in both cell types. TRPM2 overexpression remarkably augmented H2O2-induced cell death and reduction in cell viability. Furthermore, H2O2-induced cell death in both the wild-type and TRPM2-overexpressing cells was prevented by 2-APB, a TRPM2 inhibitor, and also by PJ34 and DPQ, poly(ADP-ribose) polymerase (PARP) inhibitors. Collectively, our results show that increasing the TRPM2 expression renders SH-SY5Y cells to be more susceptible to ROS-induced cell death and reinforce the notion that the TRPM2 channel plays a critical role in conferring ROS-induced cell death. It is anticipated that SH-SY5Y cells can be useful for better understanding the molecular and signaling mechanisms for ROS-induced TRPM2-mediated neurodegeneration in the pathogenesis of neurodegenerative diseases.

Orai1 is involved in leptin-sensitive cell maturation in mouse dendritic cells.

Store operated calcium entry(SOCE) is known to play a pivotal role in DCs functions including migration, maturation and antigen-presenting ability. Orai1, the major component of SOCE which mainly pairs with Stim1, is surely involved in the regulation of DCs functions. Leptin is recently found to mature DCs, we aim to evaluate the role of Orai1 in leptin-induced dendritic cells(DCs) maturation process and elucidate the mechanism. To this end, Flow cytometry and ELISA were utilized to detect the costimulatory molecule CD86 expression and IL-12 secretion, respectively. Transwell assay was used to examine DCs migration capacity. To evaluate the activity of SOCE, calcium(Ca2+) imaging was performed. Firstly, we confirmed the positive effects of leptin upon SOCE and Orai1 expression in DCs isolated from mouse bone marrow. Secondly, we showed that the effects of leptin on DCs migration and maturation are Orai1 dependent. Moreover, Janus kinase 2(Jak2) silencing inhibited leptin-induced Orai1 expression and influenced DCs functions including migration and maturation as well as IL-12 secretion. In conclusion, our results imply that leptin regulates Orai1 by activating Jak2 signaling pathway, hence facilitating DCs migration and maturation.

Reticulocalbin 3 Deficiency in Alveolar Epithelium Exacerbated Bleomycin-induced Pulmonary Fibrosis.

Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum (ER) lumen protein localized to the secretory pathway. We have reported that Rcn3 plays a critical role in alveolar epithelial type II cell maturation during perinatal lung development, but its biological role in the adult lung is largely unknown. In this study, we found marked induction of Rcn3 expression in alveolar epithelium during bleomycin-induced pulmonary fibrosis, which is most obvious in alveolar epithelial type II cells (AECIIs). To further examine Rcn3 in pulmonary injury remodeling, we generated transgenic mice to selectively delete Rcn3 in AECIIs in adulthood. Although Rcn3 deletion did not cause obvious abnormalities in the lung architecture and mechanics, the exposure of Rcn3-deleted mice to bleomycin led to exacerbated pulmonary fibrosis and reduced lung mechanics. These Rcn3-deleted mice also displayed enhanced alveolar epithelial cell (AEC) apoptosis and ER stress after bleomycin treatment, which was confirmed by in vitro studies both in primary AECIIs and mouse lung epithelial cells. Consistently, Rcn3 deficiency also enhanced ER stress and apoptosis induced by ER stress inducers, tunicamycin and thapsigargin. In addition, Rcn3 deficiency caused blunted wound closure capability of AECs, but not altered proliferation and bleomycin-induced epithelial-mesenchymal transition process. Collectively, these findings indicate that bleomycin-induced upregulation of Rcn3 in AECIIs appears to contribute to AECII survival and wound healing. These observations, for the first time, suggest a novel role of Rcn3 in regulating pulmonary injury remodeling, and shed additional light on the mechanism of idiopathic pulmonary fibrosis.

ATP-activated P2X7 receptor in the pathophysiology of mood disorders and as an emerging target for the development of novel antidepressant therapeutics.

Mood disorders are a group of psychiatric conditions that represent leading global disease burdens. Increasing evidence from clinical and preclinical studies supports that innate immune system dysfunction plays an important part in the pathophysiology of mood disorders. P2X7 receptor, belonging to the ligand-gated ion channel P2X subfamily of purinergic P2 receptors for extracellular ATP, is highly expressed in immune cells including microglia in the central nervous system (CNS) and has a vital role in mediating innate immune response. The P2X7 receptor is also important in neuron-glia signalling in the CNS. The gene encoding human P2X7 receptor is located in a locus of susceptibility to mood disorders. In this review, we will discuss the recent progress in understanding the role of the P2X7 receptor in the pathogenesis and development of mood disorders and in discovering CNS-penetrable P2X7 antagonists for potential uses in in vivo imaging to monitor brain inflammation and antidepressant therapeutics.

The role of AKT isoforms in glioblastoma: AKT3 delays tumor progression.

The growth factor receptor/PI3K/AKT pathway is an important drug target in many cancers including Glioblastoma. AKT, a key node in the pathway, has 3 isoforms, AKT1, AKT2 and AKT3. Here we investigate their role in GBM. We find each activated, ser473 phosphorylated isoform is present in some GBMs but expression patterns vary. There is a direct relationship between human GBM patient outcome and both AKT1 and AKT2 mRNA levels, but an inverse relationship with AKT3 mRNA. Furthermore, AKT3 mRNA levels were high in a less aggressive GBM subtype. Overexpressing AKT3 improves survival in a rodent model of GBM and decreases colony forming efficiency, but not growth rate, in glioma cells. Silencing AKT3 slows cell cycle progression in one cell line and increases apoptosis in another. Our studies of AKT3 substrates indicate (1) silencing both AKT2 and AKT3 reduces GSK3 phosphorylation (2) only AKT2 silencing reduces S6 phosphorylation. Since S6 phosphorylation is a marker of mTORC1 activity this indicates that AKT2 activates mTORC1, but AKT3 does not. Our results indicate AKT isoforms have different roles and downstream substrates in GBM. Unexpectedly, they indicate AKT3 delays tumor progression. Therefore strategies that inhibit AKT3 may be unhelpful in some GBM patients.

[Construction of eukaryotic expressing vector of human P2X7 and establishment of stable transfectant cell line].

OBJECTIVE: To construct eukaryotic expression vector of human P2X7gene and transfect HEK293 cells so as to establish stable HEK293 cell line. METHODS: P2X7 gene was amplified by polymerase chain reaction from the human brain P2X7 cDNA and inserted into a vector pEGFP-N1 to construct a recombinant plasmidcalled pEGFP-N1/P2X7. The correct recombinant plasmid was transfected into HEK293 cells by X-fect transfection reagent. The cell line stably expressing EGFP tagged-P2X7 gene were established by screening with G418 and fluorescence microscope. The expression levels and localization of human P2X7 in HEK293 cells was identified by flow cytometry, Western blot and laser scanning confocal microscope. RESULTS: The recombinant plasmid pEGFP-N1/P2X7 was constructed correctly and the stable HEK293 cell line expressing EGFP tagged-P2X7 fusion protein was established. Both Western blot and flow cytometry revealed the higher expression of humanP2X7 in the stably transfected HEK293 cells. Under the laser scanning confocal microscope the EGFP tagged-P2X7 fusion protein was located on the membrane of HEK293 cells. CONCLUSIONS: The eukaryotic expressing vector of pEGFP-N1/P2X7 is successfully constructed and the HEK293 cell line stably expressing P2X7-EGFP fusion protein is established which have provided solid experimental foundation for further studies on the structure and function of P2X7 ionic channel.