High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.     

Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells

Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1-34) and specifically cross-linked to a radioiodinated bPTH-(1-34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.     

High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors

The human neuronal Cys‐loop ligand‐gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high‐level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high‐level heterologous production of pure receptors in a state that supports agonist‐induced allosteric conformational changes. In a tetracycline‐inducible stable human embryonic kidney cells (HEK293S) cell line, GABA_A receptors containing α1 and β3 subunits could be expressed with specific activities of 29–34 pmol/mg corresponding to 140–170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5‐HT_3A) receptors were 49–63 pmol/mg and 245–315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3–3.0 L. Both receptor constructs had a FLAG epitope inserted at the N‐terminus and could be purified in one step after solubilization using ANTI‐FLAG affinity chromatography with yields of 30–40%. Purified receptors were functional. Binding of the agonist [³H]muscimol to the purified GABA_AR was enhanced allosterically by the general anesthetic etomidate, and purified 5‐hydroxytryptamine‐3A receptor supported serotonin‐stimulated cation flux when reconstituted into lipid vesicles.     

S1P-receptors in PC12 and transfected HEK293 cells: Molecular targets of hypotensive imidazoline I1 receptor ligands

The present study aimed at elucidating the molecular identity of the proposed “I₁-imidazoline receptors”, i.e. non-adrenoceptor recognition sites via which the centrally acting imidazolines clonidine and moxonidine mediate a major part of their effects. In radioligand binding experiments with [³H]clonidine and [³H]lysophosphatidic acid on intact, α₂-adrenoceptor-deficient PC12 cells, moxonidine, clonidine, lysophosphatidic acid and sphingosine-1-phosphate (S1P) competed for the specific binding sites of both radioligands with similar affinities. RNA interference with the rat S1P₁-, S1P₂- or S1P₃-receptor abolished specific [³H]lysophosphatidic acid binding. [³H]Clonidine binding was markedly decreased by siRNA targeting S1P₁- and S1P₃-receptors but not by siRNA against S1P₂-receptors. Finally, in HEK293 cells transiently expressing human S1P₃-receptors, sphingosine-1-phosphate, clonidine and moxonidine induced increases in intracellular calcium concentration, moxonidine being more potent than clonidine; this is in agreement with the known properties of the “I₁-imidazoline receptors”.The present results indicate that the “I₁-imidazoline receptors” mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belong to the S1P-receptor family; in particular, the data obtained in PC12 cells suggest that the I₁ imidazoline receptors represent a mixture of S1P₁- and S1P₃-receptors and/or hetero-dimers of both.     

S1P-receptors in PC12 and transfected HEK293 cells: molecular targets of hypotensive imidazoline I(1) receptor ligands

The present study aimed at elucidating the molecular identity of the proposed “I₁-imidazoline receptors”, i.e. non-adrenoceptor recognition sites via which the centrally acting imidazolines clonidine and moxonidine mediate a major part of their effects. In radioligand binding experiments with [³H]clonidine and [³H]lysophosphatidic acid on intact, ₂-adrenoceptor-deficient PC12 cells, moxonidine, clonidine, lysophosphatidic acid and sphingosine-1-phosphate (S1P) competed for the specific binding sites of both radioligands with similar affinities. RNA interference with the rat S1P₁-, S1P₂- or S1P₃-receptor abolished specific [³H]lysophosphatidic acid binding. [³H]Clonidine binding was markedly decreased by siRNA targeting S1P₁- and S1P₃-receptors but not by siRNA against S1P₂-receptors. Finally, in HEK293 cells transiently expressing human S1P₃-receptors, sphingosine-1-phosphate, clonidine and moxonidine induced increases in intracellular calcium concentration, moxonidine being more potent than clonidine; this is in agreement with the known properties of the “I₁-imidazoline receptors”.

The present results indicate that the “I₁-imidazoline receptors” mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belong to the S1P-receptor family; in particular, the data obtained in PC12 cells suggest that the I₁ imidazoline receptors represent a mixture of S1P₁- and S1P₃-receptors and/or hetero-dimers of both.     

A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells

We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPgamma). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3A and 2.4A, respectively.     

Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines

Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.     

Dex-ras1 and Serum- and Glucocorticoid-inducible Protein Kinase 1: Regulation of Expression by Dexamethasone in HEK293 Cells

The molecular and cellular basis of the psychotropic actions of adrenal corticosteroids is poorly understood. Previously, we reported that modulation of large conductance Ca²⁺-activated potassium channel (BK-channel) function by glucocorticoids can be recapitulated in human embryonic kidney293 (HEK293) cells (J Physiol 537:57, 2001). In the present paper, we examined the effect of dexamethasone on the expression of candidate mediator proteins of glucocorticoid action, dex-ras1 and serum and glucocorticoid inducible protein kinase 1 (SGK), in HEK293 cells. Dex-ras1 mRNA was readily detectable under basal conditions however, no changes of dex-ras1 mRNA expression occurred upon exposure to 100 nM of dexamethasone for 2 h. In contrast, a 2.5-fold increase of SGK mRNA was found under similar conditions. Total levels of cellular SGK protein were unaltered upon exposure to dexamethasone, but a marked increase of SGK in a Triton-X100 insoluble fraction was observed. BK-channel α-subunits could not be co-immunoprecipitated with SGK. In summary, SGK, but not dex-ras1, mRNA is rapidly induced by glucocorticoid stimulation in HEK293 cells. However, there appears to be no direct protein-protein interaction between SGK and BK-channel α-subunits. Presented to mark the 70th birthday of Professor George Fink. Special issue article in honor of George Fink.     

A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification

The iodinated Mr approximately equal to 15,000 amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (u-PA) molecule bound specifically to the cell surface of all of seven cultured human tumor cell lines studied. Cross-linking of iodinated ATF to the cell surface using a bifunctional amino-reactive reagent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed with the four cell lines studied the occurrence of a single band migrating with an Mr of 70,000-75,000, indicating complex formation with an Mr of 55,000-60,000 u-PA receptor protein (u-PA-R). In the human monocyte cell line U937 cultivated in the presence of phorbol ester, the amount of complex was strongly increased, and a fraction of the complex had a slower electrophoretic mobility. Comparison between autoradiograms of reduced and unreduced samples suggests that u-PA-R consists of one polypeptide chain. Two forms of u-PA-R, which differed with respect to affinity to concanavalin A, were identified. u-PA-R retained its ability to bind to ATF after cell lysis, and it was purified approximately 2,200-fold from biosynthetically labeled U937 cells by affinity chromatography with proenzyme u-PA coupled to Sepharose. The purified Mr 55,000-60,000 protein was specifically bound and cross-linked to u-PA, proenzyme u-PA, and ATF, but not to tissue-type plasminogen activator or other unrelated proteins.     

Expression of Cry1Ac cadherin receptors in insect midgut and cell lines

Cadherin-like proteins have been identified as putative receptors for the Bacillus thuringiensis Cry1A proteins in Heliothis virescens and Manduca sexta. Immunohistochemistry showed the cadherin-like proteins are present in the insect midgut apical membrane, which is the target site of Cry toxins. This subcellular localization is distinct from that of classical cadherins, which are usually present in cell-cell junctions. Immunoreactivity of the cadherin-like protein in the insect midgut was enhanced by Cry1Ac ingestion. We also generated a stable cell line Flp-InT-REX-293/Full-CAD (CAD/293) that expressed the H. virescens cadherin. As expected, the cadherin-like protein was mainly localized in the cell membrane. Interestingly, toxin treatment of CAD/293 cells caused this protein to relocalize to cell membrane subdomains. In addition, expression of H. virescens cadherin-like protein affects cell-cell contact and cell membrane integrity when the cells are exposed to activated Cry1Ab/Cry1Ac.     

Expression,purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-x_L, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-x_L, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-x_L fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-x_L and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-x_L at the atomic level.     

Expression and functional purification of a glycosylation deficient version of the human adenosine 2a receptor for structural studies

A glycosylation deficient (dG) version of the human adenosine 2a receptor (hA2aR) was made in Pichia pastoris strain SMD1163. Under optimal conditions, expression levels of between 8 and 12pmol receptor/mg membrane protein were obtained routinely. In a shake flask, this is equivalent to ca. 0.2mg of receptor per litre of culture. The level of functional receptor produced was essentially independent of the pH of the yeast media. In contrast to this, addition of the hA2aR antagonist theophylline to the culture media caused a twofold increase in receptor expression. A similar effect on dG hA2aR production was also observed when the induction temperature was reduced from 29 to 22 degrees C. In P. pastoris membranes, dG hA2aR had native-like pharmacological properties, binding antagonists with rank potency ZM241385>XAC>theophylline, as well as the agonist NECA. Furthermore, the receptor was made with its large (ca. 120 amino acid) C-terminal domain intact. dG hA2aR was purified to homogeneity in three steps, and its identity confirmed by electrospray tandem mass spectrometry following digestion with trypsin. The secondary structure of the entire receptor is largely (ca. 81%) alpha-helical. Purified dG hA2aR bound [(3)H]ZM241385 in a saturable manner with a B(max) of 18.1+/-0.5 nmol/mg protein, close to the theoretical B(max) value for pure protein (21.3 nmol/mg protein), showing that the receptor had retained its functionality during the purification process. Regular production of pure dG hA2aR in milligram quantities has enabled crystallisation trials to be started.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 μM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.     

Expression of receptor protein tyrosine phosphatase agr; mRNA in human prostate cancer cell lines

Receptor protein tyrosine phosphatase (RPTP) is transmembrane protein phosphatases, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTP mRNA in several normal human tissues. We further investigated the regulation of expression of RPTP mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5-dihydrotestosterone (DHT) down-regulates RPTP mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTP mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTP as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTP mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTP is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.     

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His(5)) with an NH(2)-terminal leader peptide. The 4D5 scFv-barnase-His(5) gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His(5) fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His(5) on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.     

Establishment and characterization of RNA-edited serotonin 2C receptor isoform cell models and alteration of amyloid precursor protein ectodomain secretion in HEK293 APPSwe cells

RNA editing is a mechanism for generating molecular diversity by altering the genetic code at the level of RNA. The 5-HT(2C) receptor is the only G protein-coupled receptor known to be edited. It has been reported that the non-edited 5-HT(2C) receptor stimulates secretion of the APP metabolite APP ectodomain (APPs). However, it remains unknown whether RNA-edited 5-HT(2C) receptors can also affect APPs secretion. In this study, cDNAs of five non-edited or partially/fully edited 5-HT(2C) receptor isoforms (INI, VNI, VNV, VSV and VGV) were stably transfected into HEK293APPSwe cells to detect the cell proliferation and APPs secretion. The results demonstrated that the overexpression of INI and VNI caused increased proliferation of host cells while VNV, VSV and VGV caused inverse effects (P?相似文献   

Comparative studies on the structural gene for the ribosomal protein S1 in ten bacterial species

Summary Callus protoplasts of a plastome chlorophyll-deficient mutant of tobacco, Nicotiana tabacum, were fused with mesophyll protoplasts from one of the following five sources: cms-analogs of tobacco bearing the cytoplasms of N. suaveolens, N. undulata, N. repanda, and N. plumbaginifolia, respectively, and the wild species N. glauca. In the sixth experiment, callus cells of the tobacco chlorophyll-deficient genome mutant, homozygous for the Su gene, were hybridized with mesophyll protoplasts of the plastome chlorophyll-deficient mutant of tobacco. Individual dividing heteroplasmic fusion products were isolated mechanically and cloned in microdroplets of nutrient medium. Among the regenerants in all parental combinations, though not in all clones, besides pure green and pure chlorophyll-deficient plants, numerous variegated plant forms were obtained. The variegation of cybrid and hybrid plants was connected with their heterozygocity for chloroplast DNA composition, as demonstrated by restriction analysis. In analytical crosses, the variegation was inherited maternally by part of the sexual progeny, and variegated F₁ progeny were also heterozygous for chloroplast DNA composition. As demonstrated by electron microscopic studies, the variegation is connected with the presence of mixed, i.e., heteroplastidic cells in leaves. The results obtained demonstrate that (1) upon somatic cell fusion, plastome genes are inherited biparentally in most fusion products, and (2) despite an evident mitotic segregation process, heterozygosity for plastome genes is a relatively durable state and can be revealed in cell hybrids of different specific combinations of plasmons after a great number of cell generations. In this regard the plastome cytogetes obtained by somatic cell fusion do not differ qualitatively from the cytoplasmic heterozygotes that arise as a result of the mutation process.