Soluble Glutathione S-transferase Fusion Proteins Research Articles

Prokaryotic Expression of AATF-Che1 Superfamily Gene and Its Fusion Protein Purification

Objective To construct the apoptosis antagonizing transcription factor (AATF)-Che1 recombinant prokaryotic expression vector pGEX-4T-1-AATF-Che1 in vitro,optimize prokaryotic recombinant vector induced expression conditions,and get soluble purified glutathione S-transferase(GST) fusion proteins.Methods The AATF-Che1 gene was amplificated from the pGEX-4T-1-AATF recombinant plasmid by polymerase chain reaction(PCR).The prokaryotic expression vector pGEX-4T-1 was digested with double enzymes,and recycled purpose fragments.The directional connection of AATF-Che1 gene and linearization prokaryotic expression vector pGEX-4T-1 were through the T4 ligase,to obtain recombinant protein AATF-Che1 prokaryotic expression plasmid pGEX-4T-1-AATF-Che1.Correct plasmid was transformated to escherichia coli cells BL21 (DE3),and used isopropy1β-D-1-thiogalactopyranoside (IPTG) to induce AATF-Che1 protein expression.Through exploring the influence of the IPTG concentration,induction time and temperature to the expression quantity and form of the recombinant protein,optimized the best induction conditions.Using glutathione sepharose beads purificated soluble GST fusion protein,at last to identificate of the protein by Western blot (WB) mothd.Results The results of enzyme digestion,PCR identification and sequencing were correct,and the prokaryotic expression plasmid was successfully constructed.The electrophoresis showed that a new protein band appear at about 65 kD,which was the fusion protein of AATF-Che1 and GST.The fusion protein was no obvious change with different induction time and IPTG concentration at 37℃ or 16℃.The recombinant protein mainly existed in the form of inclusion body at 37 ℃,while soluble from in the supernatant at 16℃.Soluble purificated GST fusion protein was obtained,and by WB method identified antigenicity of fusion protein was good.Conclusions The prokaryotic expression plasmid was successfully constructed in vitro.The optimized recombinant prokaryotic expression conditions were found and purificated GST fusion protein was obtained.It maybe lay the foundation for the further study on the role of AATF-Che1 in tumor. Key words: AATF-Che1 superfamily domain; Prokaryotic expression; Fusion protein purification

Improved recovery of active GST-fusion proteins from insoluble aggregates: solubilization and purification conditions using PKM2 and HtrA2 as model proteins

The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1:200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.

Preparation of Soluble GST Fusion Proteins

A wide variety of plasmid vectors are commercially available for the production of fusion proteins in bacterial cells. Most are also designed to incorporate a tag that allows affinity purification of the expressed fusion protein from bacterial cell extracts. The most commonly used are vectors that incorporate a portion of the glutathione-S-transferase (GST) enzyme that is able to bind to immobilized glutathione and vectors that use a polyhistidine tag which binds immobilized nickel ions with high affinity. This protocol describes preparation of a soluble GST fusion protein, isolated using glutathione agarose beads.

Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts.

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil.

BackgroundThe naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil.MethodsSeven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection.ResultsThe frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals.ConclusionThis study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.

The Expanded Octarepeat Domain Selectively Binds Prions and Disrupts Homomeric Prion Protein Interactions

Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrPC) into the pathogenic conformer PrPSc, which subsequently induces further conversion in an autocatalytic manner. Apparently, an expanded octarepeat domain directs folding of PrP toward the PrPSc conformation and initiates a self-replicating conversion process. Here, based on three main observations, we have provided a model on how altered molecular interactions between wild-type and mutant PrP set the stage for familial Creutzfeldt Jakob disease with octarepeat insertions. First, we showed that wild-type octarepeat domains interact in a copper-dependent and reversible manner, a "copper switch." This interaction becomes irreversible upon domain expansion, possibly reflecting a loss of function. Second, expanded octarepeat domains of increasing length gradually form homogenous globular multimers of 11-21 nm in the absence of copper ions when expressed as soluble glutathione S-transferase fusion proteins. Third, octarepeat domain expansion causes a gain of function with at least 10 repeats selectively binding PrPSc in a denaturant-resistant complex in the absence of copper ions. Thus, the combination of both a loss and gain of function profoundly influences homomeric interaction behavior of PrP with an expanded octarepeat domain. A multimeric cluster of prion proteins carrying expanded octarepeat domains may therefore capture and incorporate spontaneously arising short-lived PrPSc-like conformers, thereby providing a matrix for their conversion.

Ileal Bile Acid-binding Protein, Functionally Associated with the Farnesoid X Receptor or the Ileal Bile Acid Transporter, Regulates Bile Acid Activity in the Small Intestine

Bile acids secreted in the small intestine are reabsorbed in the ileum where they activate the nuclear farnesoid X receptor (FXR), which in turn stimulates expression of the ileal bile acid-binding protein (I-BABP). We first hypothesized that I-BABP may negatively regulate the FXR activity by competing for the ligands, bile acids. Reporter assays using stable HEK293 cell lines expressing I-BABP revealed that I-BABP enhances rather than attenuates FXR activity. In these cells I-BABP localizes predominantly in the cytosol and partially in the nucleus, a distribution that does not shift in response to FXR expression. In vitro binding assays reveal that recombinant I-BABP is able to bind 35S-labeled FXR and that chenodeoxycholic acid (CDCA) stimulates this interaction modestly. When FLAG-tagged FXR was expressed in stable cells, the FXR.I-BABP complex in the nuclear extracts was more efficiently immunoprecipitable with anti-FLAG antibodies in the presence of CDCA. These results indicate that I-BABP stimulates FXR activity through a mutual interaction augmented by bile acids. When stable cells were transfected with an expression plasmid of the ileal bile acid transporter 14(IBAT) essential for the reabsorption of conjugated bile acids, the C-labeled conjugated bile acid, glycocholic acid, was more efficiently imported via IBAT in the presence than absence of I-BABP, whereas no change was observed in 14C-labeled CDCA uptake, which is independent of IBAT. Immunofluorescent staining analysis revealed that these two proteins co-localize in the vicinity of the plasma membrane in stable cells. Taken together, the current data provide the first evidence that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, stimulating FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.

Multivalent Interactions of Calcium/Calmodulin-dependent Protein Kinase II with the Postsynaptic Density Proteins NR2B, Densin-180, and α-Actinin-2

Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.

Distal Substrate Interactions Enhance Plasmepsin Activity

Plasmepsin II (PM II) is an aspartic protease active in hemoglobin (Hb) degradation in the protozoan parasite Plasmodium falciparum. A fluorescence-quenched octapeptide substrate based on the initial hemoglobin cleavage site is recognized well by PM II. C-terminal extension of this peptide has little effect, but N-terminal extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition. This inhibition, but not the rate stimulation, depends on the presence of a DABCYL group on the peptide N terminus. Using site-directed mutagenesis, we have identified PM II residues that interact with N-terminal amino acids of peptide substrates. The same residues influence degradation of Hb by PM II. Cathepsin E (CatE), a related mammalian aspartic protease, is also stimulated by N-terminally extended substrates. This suggests that distal substrate interactions as far out as P6 may be a general property of aspartic proteases and may be important in substrate and inhibitor recognition. Although PM II and CatE are similar in their ability to cleave Hb-based peptides and Hb alpha-chains, CatE is not able to degrade native Hb, which is a substrate for PM II. Based on these results, we propose that PM II may have the special feature of being a Hb denaturase.

Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli.

We developed an assay to directly measure the ligand binding properties of the cloned human erythropoietin receptor (EpoR). The cDNA encoding the extracellular domain of the human EpoR was amplified by polymerase chain reaction and ligated into the prokaryotic expression vector pGEX3X. Synthesis in Escherichia coli was induced and a soluble glutathione S-transferase fusion protein, EREx, was purified by erythropoietin affinity chromatography. Purified EREx was bound to GSH agarose beads and used in a solid phase ligand binding assay. Specific binding of 125I-erythropoietin to EREx beads was demonstrated. A single affinity class (Kd = 1.5 nM) of the binding site was evident on Scatchard analysis. The Kd of this site is quantitatively equivalent to that of the "low" affinity cellular binding site. Kinetic analysis of ligand binding to EREx revealed both the on and off rates to be rapid, with t1/2 of 60 and 40 s, respectively. EREx ligand binding exhibits no obvious metal ion dependence or cross-competition by other hemopoietins. Antibodies to EREx block the binding of erythropoietin to the cellular EpoR. We conclude that the 66-kDa EpoR protein is capable of specific ligand binding and that no covalent modifications or associated molecules are required for this interaction. We speculate that the "high" affinity cellular binding site (Kd less than 0.2 nM) results from the interaction of the EpoR with another molecule, either additional EpoR or associated subunits, that decreases the ligand off rate.