Glutathione S-transferase Fusion Protein Research Articles

Abiotic stress response in yeast and metal-binding ability of a type 2 metallothionein-like protein (PutMT2) from Puccinellia tenuiflora.

Metallothioneins are low-molecular weight and cysteine-rich metal-binding proteins that play predominant cellular roles in the scavenging of reactive oxygen species and in mediating metal metabolism. To evaluate the role of a type-2 metallothionein-like gene from Puccinellia tenuiflora (PutMT2), the gene was over-expressed in yeast, and growth was assessed under a variety of abiotic stress conditions including peroxide (H2O2), salinity (NaCl and NaHCO3), and metal stress. PutMT2 overexpression in yeast improved the tolerance of cells to H2O2, NaCl, NaHCO3, Zn(2+), Fe(2+), Fe(3+), Cd(2+), Cr(6+), and Ag(+), but increased the sensitivity of cells to Mn(2+), Co(2+), Cu(2+), and Ni(2+) compared with control cells. PutMT2 was then expressed in Escherichia coli BL21as a glutathione S-transferase (GST) fusion protein (GST-PutMT2), and the metal-binding ability of GST-PutMT2 was analyzed and compared with GST alone using inductively coupled plasma atomic emission spectroscopy. Results showed that PutMT2 could bind to Cr, Cd, Co, Ag, Ba, Pb, Mn, Zn, Fe, Cu, P, Al, and Mg, but not Ni and As. There was no evidence to suggest that PutMT2 exhibited a specific or selective binding tendency to any individual metal ion. PutMT2 protein bound to Zn, Na, and Cu in vivo, perhaps with the highest affinity for Cu. Taken together, our results suggest that PutMT2 protein could play an important role in improving metal tolerance by metal binding in yeast. However, additional studies are required to confirm these results and to clarify the function of PutMT2 in transgenic plants.

Immunisation with bacterial expressed VP2 and VP5 of bluetongue virus (BTV) protect α/β interferon-receptor knock-out (IFNAR−/−) mice from homologous lethal challenge

BTV-4 structural proteins VP2 (as two domains: VP2D1 and VP2D2), VP5 (lacking the first 100 amino acids: VP5Δ1–100) and full-length VP7, expressed in bacteria as soluble glutathione S-transferase (GST) fusion-proteins, were used to immunise Balb/c and α/β interferon receptor knock-out (IFNAR−/−) mice. Neutralising antibody (NAbs) titres (expressed as log10 of the reciprocal of the last dilution of mouse serum which reduced plaque number by ≥50%) induced by the VP2 domains ranged from 1.806 to 2.408 in Balb/c and IFNAR−/− mice.The immunised IFNAR−/− mice challenged with a homologous live BTV-4 survived and failed to develop signs of infection (ocular discharge and apathy). Although subsequent attempts to isolate virus were unsuccessful (possibly reflecting presence of neutralising antibodies), a transient/low level viraemia was detected by real time RT-PCR. In contrast, mice immunised with the two VP2 domains with or without VP5Δ1–100 and VP7, then challenged with the heterologous serotype, BTV-8, all died by day 7 post-infection.We conclude that immunisation with bacterially-expressed VP2 domains can induce strong serotype-specific NAb responses. Bacterial expression could represent a cost effective and risk-free alternative to the use of live or inactivated vaccines, particularly if viruses prove to be difficult to propagate in cell culture (like BTV-25). A vaccine based on bacterially expressed VP2 and VP5 of BTV is also DIVA-compatible.

Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378-455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.

A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif

A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [¹⁵N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at -80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production.

Participation of the extracellular domain in (pro)renin receptor dimerization

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.

Biosynthesis of a potentially functional polypeptide derived from silk fibroin.

In order to understand the relationship between sequences and biological functions of RGD-containing wild silkworm silk fibroin, it is important to purify the basic RGD-containing motif in large quantities. In this study, a gene monomer encoding RGD-contained motif GSGAGGRGDGGYGSGSS (-RGD-) derived from Antheraea pernyi (the same in Antheraea yamamai) was designed and cloned. (-RGD-)n in various degrees of polymerizations was obtained by gene monomer doubling-extension and expression. Two glutathione-S-transferase (GST)-tagged fusion proteins GST-(-RGD-)12 and GST-(-RGD-)24 were successfully expressed in Escherichia coli (E. coli) BL21. The fusion proteins were isolated and purified by GST affinity chromatography, and the polypeptides (-RGD-)12 and (-RGD-)24 were cleaved from GST fusion proteins by thrombin digestion. Two-dimensional electrophoresis and amino acid composition analysis were performed to confirm the identity of the engineered polypeptides. Results indicated that this technology reliably obtained expected polypeptides (-RGD-)n for future research on structure and functions.

Identification and Characterization of a <i>Trypanosoma congolense</i> 46 kDa Protein as a Candidate Serodiagnostic Antigen

ABSTRACTTrypanosoma congolense is a major livestock pathogen in Africa, causing large economic losses with serious effects on animal health. Reliable serodiagnostic tests are therefore urgently needed to control T. congolense infection. In this study, we have identified one T. congolense protein as a new candidate serodiagnostic antigen. The 46.4 kDa protein (TcP46, Gene ID: TcIL3000.0.25950) is expressed 5.36 times higher in metacyclic forms than epimastigote forms. The complete nucleotide sequences of TcP46 contained an open reading frame of 1,218 bp. Southern blot analysis indicated that at least two copies of the TcP46 gene were tandemly-arranged in the T. congolense genome. The recombinant TcP46 (rTcP46) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Western blot analysis and confocal laser scanning microscopy revealed that the native TcP46 protein is expressed in the cytoplasm during all life-cycle stages of the parasite. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on rTcP46 detected the specific antibodies as early as 8 days post-infection from mice experimentally infected with T. congolense. No cross-reactivity was observed in the rTcP46-based ELISA against serum samples from cattle experimentally infected with Babesia bigemina, B. bovis and Anaplasma marginale. These results suggest that rTcP46 could be used as a serodiagnostic antigen for T. congolense infection.

An alternative easy method for antibody purification and analysis of protein–protein interaction using GST fusion proteins immobilized onto glutathione–agarose

Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.

Identification and Characterization Of a Potent FLT3 Inhibitor

Acute myeloid leukemia (AML) is a malignant myeloid disorder for which there is no effective treatment. Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in AML patients. This makes FLT3 an attractive therapeutic target. Currently, several potent FLT3 inhibitors have been developed. However, their clinical efficacy is limited largely due to their poor effectiveness toward the FLT3-D835 mutants which are often present in AML or acquired after treatment of FLT3-ITD-positive AML with tyrosine kinase inhibitors. Needless to say, more potent FLT3 inhibitors targeting both FLT-ITD and FLT3-D835 mutants are needed. In addition, combinations of tyrosine kinase inhibitors with drugs targeting other signaling pathways represent a new trend in anti-cancer drug development.To establish an effective kinase assay for FLT3 inhibitor screening, we generated a protein substrate designated GST-FLT3S which was expressed in E. coli cells as a glutathione S-transferase fusion protein. The protein substrate together with recombinant proteins containing the catalytic domain of wild type and mutant forms of FLT3 expressed in baculovirus was used in biochemical screening of inhibitors. Several potent inhibitors were obtained. Importantly, one of the inhibitors with an oxindole core structure inhibited FLT3 and D835 FLT3 mutants equally well with nanomolar IC50 values. We further analyzed the potency of the inhibitor by performing cell-based assays. The cells used included FLT3-ITD-positive cell line MV-4-11 and an EPO-dependent erythroleukemia cell line transformed by retrovirus mediated expressions of FLT3-ITD and FLT3-D835 mutants. At nanomolar concentrations, the inhibitor blocked growth factor signaling and effectively caused apoptosis and cell cycle arrest. It showed significant advantage over the current available FLT3 inhibitor, sorafenib.Loss-of-function mutations of tumor suppressor p53 are common in solid tumors but relatively rare in AML although its expression is often suppressed. This makes p53 a potential target for anti-AML drug development. We employed MDM2 inhibitor nutlin-3 which blocks the degradation of p53. Importantly, at sub-nanomolar concentrations, FLT3 inhibitors and nutlin-3 synergistically inhibited growth of cells containing FLT3-ITD or FLT3-D835 mutants.Altogether, we developed an effective substrate for screening of FLT3 inhibitors and identified one compound with high potency toward both FLT3-ITD and FLT3-D835. We further demonstrated that targeting FLT3 and p53 simultaneously greatly increases drug potency. Disclosures:No relevant conflicts of interest to declare.

Efficient expression of truncated recombinant cadmium-metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis in Escherichia coli

Truncated recombinant metallothionein GST-fusion protein has been successfully expressed in Escherichia coli. The previously identified novel Cd-inducible metallothionein (TMCd1) gene from the locally isolated ciliate, Tetrahymena tropicalis lahorensis, was inserted into a pET-41a vector, in frame with a sequence encoding an N-terminal glutathione-S-transferase (GST) tail. Truncated recombinant GST fusion protein has been purified by affinity column chromatography using glutathione sepharose. After enzymatic cleavage of GST tail with enterokinase, the truncated TMCd1 MT shows molecular weight of 11.5kDa, corresponding to the expected value. This is the first successful report of expression of cadmium metallothionein gene of a ciliate, T. t. lahorensis, reported from this part of the world, in E. coli. This study will further help in characterization of metallothionein protein of this ciliate.

Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E. coli

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. The goal of this study was to clone, efficiently express and purify the ecdysteroid-regulated protein (ERP) in the form of a GST fusion protein. The mature peptide-coding cDNA fragment was extracted from Chinese mitten crap (Eriocheir sinensis), and then after using PCR to obtain the open reading frame, a recombinant plasmid designated pGEX-4T-1_ERP was successfully generated and showed to efficiently express the ERP fusion protein as determined by SDS-PAGE. The resulting expressed protein was successfully purified by a combination of affinity and conventional chromatographic methods. After purification, the recombinant protein showed the expected size of 41kDa on SDS-PAGE gels which was further confirmed by mass spectrometry and western blotting. Purification of recombinant protein was achieved by fast protein liquid chromatography. About 2.4mg/l recombinant protein with purity more than 80% was obtained.

Characterization of uridine diphosphate-sugar pyrophosphorylase from Populus deltoids

Nucleotide sugars serve as sugar donors for the biosynthesis of various cell components including cell wall, glycoproteins, and small molecules. Among them, uridine diphosphate (UDP)-glucose is one of main nucleotide sugars that serve as a substrate for the synthesis of other UDP-sugars. UDP-sugar pyrophosphorylase (USPase) mediates the formation of UDP-glucose from uridine triphosphate (UTP) and glucose-1-phosphate. A USPase, PdUSPase was cloned from Populus deltoids and expressed in Escherichia coli as glutathione Stransferase fusion protein. The purified recombinant PdUSPase catalyzed the reaction for the formation of UDP-glucose from glucose-1-phosphate and UTP, and for the formation of UDP-galactose from galactose-1-phosphate and UTP. However, the enzyme did not show any activity toward mannose-1-phosphate and UTP. These results indicate that PdUSPase belonging in UGPase A in phylogenetic analysis is the first UDP-glucose synthesizing enzyme showing a discrepancy between phylogenetic analysis and substrate range. E. coli complementation was also carried out to confirm the function of PdUSPase using E. coligalU mutant, which was mutated in UTP glucose-1-phosphate uridyltransferase. The galU mutant was transformed with the PdUGTase gene and a flavonoid glucosyl-transferase gene, AtUGT78D2. The resulting transformant was able to convert quercetin into quercetin 3-O-glucose similarly to that by the wild type E. coli strain harboring AtUGT78D2. These results indicated that PdUSPase catalyzed the formation of UDP-glucose from UTP and glucose-1-phosphate.

Species-Specific and Cross-Reactive IgG1 Antibody Binding to Viral Capsid Protein 1 (VP1) Antigens of Human Rhinovirus Species A, B and C

BackgroundHuman rhinoviruses (HRV) are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects.MethodsRecombinant polypeptides of viral capsid protein 1 (VP1) from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST) fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults.ResultsMost adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P<0.0001). A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies.ConclusionsHigh titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined.

High-yield production of the VP1 structural protein epitope from serotype O foot-and-mouth disease virus in Escherichia coli

For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25°C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.

A recombinant carboxy‐terminal domain of alpha‐toxin protects mice against Clostridium perfringens

Clostridium perfringens alpha-toxin (CP, 370 residues) is one of the main agents involved in the development of gas gangrene. In this study, the immunogenicity and protective efficacy of the C-terminal domain (CP251-370) of the toxin and phospholipase C (PLC; CB, 372 residues) of Clostridum bifermentans isolated from cases of clostridium necrosis were examined. The recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins. Antibodies that cross-reacted with alpha-toxin were produced after immunization with recombinant proteins including GST-CP251-370, GST-CP281-370, GST-CP311-370, CB1-372 and GST-CB251-372. Anti-GST-CP251-370, anti-GST-CP281-370 and anti-GST-CP311-370 sera neutralized both the PLC and hemolytic activities of alpha-toxin, whereas anti-CB1-372 and anti-GST-CB251-372 weakly neutralized these activities. Immunization with GST-CP251-370 and GST-CP281-370 provided protection against the lethal effects of the toxin and C. perfringens type A NCTC8237. Partial protection from the toxin and C. perfringens was elicited by immunization with GST-CP311-370 and CB1-372. GST-CP251-370 and GST-CP281-370 are promising candidates for vaccines for clostridial-induced gas gangrene.

Overexpression of Peanut Diacylglycerol Acyltransferase 2 in Escherichia coli

Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14’ using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a–GST, or AhDGAT2b–GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a–GST and AhDGAT2b–GST proteins increased the sizes of the host cells by 2.4–2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a–GST and AhDGAT2a–GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

The interplay between RPGR, PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo

Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR-interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo-loaded PDEδ and exposes the Arl2/Arl3-binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo-loaded PDEδ and Arl3 to release lipidated cargo into cilia.

Expression and purification of GST-FHL2 fusion protein

Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.

Gene Cloning and Characterization of the Protein Encoded by theNeospora caninumBradyzoite-Specific Antigen GeneBag1

Neospora caninum is an Apicomplexan parasite that causes repeated abortion and stillbirth in cattle. The aim of this study was to clone the gene encoding the N. caninum orthologue (NcBAG1) of the Toxoplasma gondii bradyzoite-specific protein TgBAG1 and characterize its expression pattern in the parasite. Isolation of the full-length 684-bp gene revealed that it shared 78.3% sequence similarity with TgBAG1. NcBAG1 encodes a predicted protein of 227 amino acids with 80.3% similarity to TgBAG1. A putative signal peptide sequence and an invariant GVL motif characteristic of small heat-shock proteins were identified in the predicted N. caninum amino acid sequence. We expressed the NcBAG1 gene as a recombinant glutathione S-transferase fusion protein (rNcBAG1) in Escherichia coli and used the purified 60 kDa protein to obtain a monoclonal antibody (Mab). rNcBAG1 reacted to Mabs specific for NcBAG1 and TgBAG1. No reaction between the NcBAG1 Mab and N. caninum tachyzoites was observed. Although the predicted molecular mass of NcBAG1 is 25 kDa, Western blot analysis of parasite lysates using the NcBAG1 Mab revealed a cross-reactive protein of approximately 30 kDa. Additionally, immunofluorescence assays using the tachyzoite-specific Mab for NcSAG1 and the bradyzoite-specific Mab for TgBAG1 or NcSAG4 revealed NcBAG1-specific expression in bradyzoites in cultures exposed to sodium nitroprusside, a reagent that increases the frequency of bradyzoites. Interestingly, the NcBAG1 protein was identified in the cytoplasm of the bradyzoite-stage parasites. This preliminary analysis of the NcBAG1 gene will assist investigations into the role of this protein in N. caninum .

Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C-terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK-293) cells. Under basal conditions, MOPr is phosphorylated on Ser(363) and Thr(370), while in the presence of morphine or [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser(356) , Thr(357) and Ser(375). Using N-terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C-terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein-coupled receptor kinase 2 (GRK2) phosphorylates Ser(375), protein kinase C (PKC) phosphorylates Ser(363), while CaMKII phosphorylates Thr(370). Phosphorylation of the GST fusion protein of the C-terminal tail of MOPr enhanced its ability to bind arrestin-2 and -3. Hence, our study identifies both the basal and agonist-stimulated phospho-acceptor sites in the C-terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.