GST Fusion Protein Research Articles

Investigating Functional and Folding Stability of an Engineered E. coli L-asparaginase Harboring Y176F/S241C Mutations.

L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from Escherichia coli bacteria with Y176F/S241C mutations. The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in E. coli, and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler's reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme. The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218 (mM/min), 273.9 (min-1), 237.8 (IU/mg) and 19.62 (mM-1 min-1), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively. The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications.

Role of Recognition MicroRNAs in Hemaphysalis longicornis and Theileria orientalis Interactions.

Ticks are an important type of pathogen transmission vector, and pathogens not only cause serious harm to livestock but can also infect humans. Because of the roles that ticks play in disease transmission, reducing tick pathogen infectivity has become increasingly important and requires the identification and characterization of these pathogens and their interaction mechanisms. In this study, we determined the miRNA expression profile of Hemaphysalis longicornis infected with Theileria orientalis, predicted the target genes of miRNAs involved in this infection process, and investigated the role of miRNA target recognition during host-pathogen interactions. The results showed that longipain is a target gene of miR-5309, which was differentially expressed at different developmental stages and in various tissues in the control group. However, the miR-5309 level was reduced in the infection group. Analysis of the interaction between miRNA and the target gene showed that miR-5309 negatively regulated the expression of the longipain protein during the infection of H. longicornis with T. orientalis. To verify this inference, we compared longipain with the blocking agent orientalis. In this study, the expression of longipain was upregulated by the inhibition of miR-5309 in ticks, and the ability of the antibody produced by the tick-derived protein to attenuate T. orientalis infection was verified through animal immunity and antigen-antibody binding tests. The results showed that expression of the longipain + GST fusion protein caused the cattle to produce antibodies that could be successfully captured by ticks, and cellular immunity was subsequently activated in the ticks, resulting in a subtractive effect on T. orientalis infection. This research provides ideas for the control of ticks and tickborne diseases and a research basis for studying the mechanism underlying the interaction between ticks and pathogens.

Monitoring of enzymatic cleavage reaction of GST-fusion protein on biolayer interferometry sensor

Monitoring of enzymatic cleavage reaction of GST-fusion protein on biolayer interferometry sensor

Junctional Adhesion Molecule a (JAM-A) Associates with CD36 upon Platelet Activation and Cytoskeleton in a Phosphorylation-Dependent Manner to Promote Platelet-Platelet Contacts

Background: Platelets play a fundamental role inmaintaining hemostasis. Stability of hemostatic plug is crucial for arresting blood loss during vascular breach. JAM-A is a member of the immunoglobulin superfamily that restricts α IIbβ 3 to its low affinity state by recruiting CSK to the α IIbβ 3-c-Src complex in resting platelets. Agonist-induced inside-out signaling causes JAM-A to dissociate from the α IIbβ 3-Src complex, thus facilitating activation of α IIbβ 3 and outside-in signaling. However, the function of JAM-A in the process of hemostasis is poorly understood. Aim: To evaluate the role of JAM-A in activated platelets and on hemostasis. Methods: Washed human platelets were used in these studies. Full-length cytoplasmic domain of JAM-A (cyto JAM-A) and JAM-A cytoplasmic domain lacking C-terminal PDZ-domain-binding motif (ΔPDZ cyto JAM-A) were expressed as GST-fusion proteins in bacteria using pGEX4T-1 vector. GST-fusion proteins were purified using glutathione beads. Pull-down assays were performed by incubating glutathione GST-fusion proteins beads with lysates of washed human platelets (4×10 8/mL). GST beads alone without the fusion proteins were used as a control. Immunoprecipitation studies were performed using washed human platelets (4x10 8/mL) lysed with CHAPS lysis buffer and precleared by using Protein G Sepharose beads. Specific primary antibodies and isotype specific IgGs (control) were used for immunoprecipitation followed by western blotting. To identify protein complexes two-dimensional blue-native polyacrylamide gel electrophoresis was performed in which protein complexes were first separated based on their size (1 st dimension) and individual proteins in each complex were separated using SDS PAGE (2 nd dimension). JAM-A S 284 phosphorylation was detected using anti-Phospho-S 284. Rap1 activation assay was performed by pull-down GTP-bound Rap1 following the manufacturer's instructions. Results: CD9 and α IIbβ 3 co-immunoprecipitated with JAM-A from resting platelet lysates suggesting that CD9 is a part of the JAM-A/α IIbβ 3 complex. In a pull-down assay recombinant full-length JAM-A cytoplasmic tail fused with GST, but not JAM-A tail lacking the PDZ domain motif fused with GST pull-down CD9 and α IIbβ 3 from resting platelet lysates suggesting that PDZ-domain binding motif of JAM-A is responsible for its interaction with CD9/α IIbβ 3 complex. Interestingly, platelet activation resulted in the dissociation of JAM-A from the CD9/α IIbβ 3 complex as seen by loss of interaction between JAM-A and CD9/α IIbβ 3 in co-immunoprecipitation and blue-native PAGE assays. It is reported that platelet activation results in rapid phosphorylation of JAM-A on S 284 in a PKC-dependent manner. We found that S 284 phosphorylated JAM-A associates with CD36 and this complex moves to the Triton X-100 insoluble cytoskeletal fraction upon platelet activation suggesting that phosphorylation of JAM-A may assist its localization to cytoskeleton. Interestingly, we found that induction of conformational change in α IIbβ 3 using Mn 2+ (10μM) activates JAM-A S 284 phosphorylation. Furthermore, Mn 2+ also induced a robust activation of Rap1, a key signaling molecule involved in junctional assembly. Both JAM-A phosphorylation and Rap1 activation were significantly ( P<0.05) inhibited by a pan PKC inhibitor, bisindolylmaleimide (10μM) suggesting that S 284 phosphorylated JAM-A/CD36 complex may support junctional assembly within the platelet plug. Conclusion: JAM-A, through its cytoplasmic domain, associates with α IIbβ 3 and CD9 in resting platelets. Upon platelet activation JAM-A rapidly dissociates from this complex and is phosphorylated on S 284. Phosphorylated JAM-A then associates with CD36 and cytoskeleton leading to activation of Rap1, a protein important in junctional assembly between platelets within a hemostatic plug.

The main protease 3CLpro of the SARS-CoV-2 virus: how to turn an enemy into a helper.

Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists.

Characterization of AtBAG2 as a Novel Molecular Chaperone

Bcl-2-associated anthanogene (BAG) family proteins regulate plant defense against biotic and abiotic stresses; however, the function and precise mechanism of action of each individual BAG protein are not yet clear. In this study, we investigated the biochemical and molecular functions of the Arabidopsis thaliana BAG2 (AtBAG2) protein, and elucidated its physiological role under stress conditions using mutant plants and transgenic yeast strains. The T-DNA insertion atbag2 mutant plants were highly susceptible to heat shock, whereas transgenic yeast strains ectopically expressing AtBAG2 exhibited outstanding thermotolerance. Moreover, a biochemical analysis of GST-fused recombinant proteins produced in bacteria revealed that AtBAG2 exhibits molecular chaperone activity, which could be attributed to its BAG domain. The relevance of the molecular chaperone function of AtBAG2 to the cellular heat stress response was confirmed using yeast transformants, and the experimental results showed that overexpression of the AtBAG2 sequence encoding only the BAG domain was sufficient to impart thermotolerance. Overall, these results suggest that the BAG domain-dependent molecular chaperone activity of AtBAG2 is indispensable for the heat stress response of Arabidopsis. This is the first report demonstrating the role of AtBAG2 as a sole molecular chaperone in Arabidopsis.

Barley metallothionein isoforms, MT2b2 and MT4, differentially respond to photohormones in barley aleurone layer and their recombinant forms show different affinity for binding to zinc and cadmium.

Metallothioneins (MTs) are metal-binding proteins that have important roles in the homeostasis of heavy metals. In this study, the two MT genes was studied in response to phytohormones using the barley aleurone layer as a kind of model system. The aleurone layer was isolated from barley embryo-less half grains and was incubated for 24h with different phytohormones. Based on the results the genes encoding HvMT2b2 and HvMT4 were down-regulated through gibberellic acid (GA), while they were and up-regulated through salicylic acid (SA). Despite this, these two genes were differentially expressed to other hormones. Furthermore, the proteins HvMT2b2 and HvMT4 were heterologous expressed as GST-fusion proteins in E. coli. The HvMT4 and HvMT2b2 heterologous expression in E. coli gives rise to 10- and 3-fold improvements in the accumulation capacity for Zn2+, respectively. Whereas the transgenic E. coli strain that expresses HvMT2b2 could accumulate Cd2+ three-fold higher than control. The expression of HvMT4 did not affect the accumulation of Cd2+. HvMT4 which is known as seed-specific isoform seems to be able to bind to Zn2+ with good affinity and cannot bind Cd2+. In comparison, HvMT2b2 was able to bind both Zn2+ and Cd2+. Therefore HvMT4 could serve a noteworthy role in zinc storage in barley seeds. The expression of HvMT4 is induced by SA 30-fold, concerning the untreated aleurone layer. Such results could provide good insights for the assessment of the effects of phytohormones in the molecular mechanism involved in essential metal storage in cereal seeds.

E3 ubiquitin ligase MAGI3 degrades c-Myc and acts as a predictor for chemotherapy response in colorectal cancer

BackgroundRecurrence and chemoresistance constitute the leading cause of death in colorectal cancer (CRC). Thus, it is of great significance to clarify the underlying mechanisms and identify predictors for tailoring adjuvant chemotherapy to improve the outcome of CRC.MethodsBy screening differentially expressed genes (DEGs), constructing random forest classification and ranking the importance of DEGs, we identified membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) as an important gene in CRC recurrence. Immunohistochemical and western blot assays were employed to further detect MAGI3 expression in CRC tissues and cell lines. Cell counting kit-8, plate colony formation, flow cytometry, sub-cutaneous injection and azoxymethane plus dextran sulfate sodium induced mice CRC assays were employed to explore the effects of MAGI3 on proliferation, growth, cell cycle, apoptosis, xenograft formation and chemotherapy resistance of CRC. The underlying molecular mechanisms were further investigated through gene set enrichment analysis, quantitative real-time PCR, western blot, co-immunoprecipitation, ubiquitination, GST fusion protein pull-down and immunohistochemical staining assays.ResultsOur results showed that dysregulated low level of MAGI3 was correlated with recurrence and poor prognosis of CRC. MAGI3 was identified as a novel substrate-binding subunit of SKP1-Cullin E3 ligase to recognize c-Myc, and process c-Myc ubiquitination and degradation. Expression of MAGI3 in CRC cells inhibited cell growth, promoted apoptosis and chemosensitivity to fluoropyrimidine-based chemotherapy by suppressing activation of c-Myc in vitro and in vivo. In clinic, the stage II/III CRC patients with MAGI3-high had a significantly good recurrence-free survival (~ 80%, 5-year), and were not necessary for further adjuvant chemotherapy. The patients with MAGI3-medium had a robustly good response rate or recurrence-free survival with fluoropyrimidine-based chemotherapy, and were recommended to undergo fluoropyrimidine-based adjuvant chemotherapy.ConclusionsMAGI3 is a novel E3 ubiquitin ligase by degradation of c-Myc to regulate CRC development and may act as a potential predictor of adjuvant chemotherapy for CRC patients.Graphical

SARS-CoV-2 Membrane protein-specific antibodies from critically ill COVID-19-infected individuals are potent stimulators of NK cell activation

Introduction. The M glycoprotein is the most abundant structural protein of the SARS-CoV-2 particle and is one of the key components for virion assembly and morphogenesis. It is a major source of peptide antigens driving T-cell responses and previous studies have shown that most patients make antibodies against its N-terminal domain.Nevertheless, no studies of the function of M-specific antibodies have been reported. Materials and Methods. We expressed the first 23 amino-acids of the M protein as a GST-fusion protein in E.coli. Sera from 37 COVID-19 patients (20 of them critically ill, admitted to ICU; 17 of them with mild disease) were assayed in a standard ELISA assay to detect reactive antibodies. The neutralization capacity of M-specific antibodies was assessed in neutralization experiments against SARS-CoV-2 virus using polyclonal antibodies purified by affinity chromatography from patient sera, while the ability of the antibodies in patients’ sera to activate Natural Killer cells was measured in an ELISA-based assay in which antigen-bound antibodies were incubated with PBMC and NK cell activation was detected by flow cytometry. Results. We could detect M-specific antibodies in most of the patients, and they could discriminate between infected and non-infected individuals with around 90% efficacy. No significant difference in the quantity of the antibody response was found between mild and critical patients. No polyclonal M-specific antibodies showed neutralization activity in the in vitro system used. Nevertheless, when PBMC from healthy donors were incubated with patients’ M- specific antibodies bound to the GST-M fusion protein in an ELISA plate, NK cells were significantly activated, as measured by LAMP1+ degranulation and cytokine (MIP1β) production. Conclusion. We show that most SARS-CoV-2 infected individuals produce specific antibodies against a surfaceexposed epitope at the N-terminus of M glycoprotein. Although these antibodies do not appear to neutralize the virus efficiently, they are able to mediate Fc/FcγR interactions to drive the activation of NK cells.

Loss of RPS27a expression regulates the cell cycle, apoptosis, and proliferation via the RPL11-MDM2-p53 pathway in lung adenocarcinoma cells

BackgroundDepletion of certain ribosomal proteins induces p53 activation, which is mediated mainly by ribosomal protein L5 (RPL5) and/or ribosomal protein L11 (RPL11). Therefore, RPL5 and RPL11 may link RPs and p53 activation. Thus, this study aimed to explore whether RPs interact with RPL11 and regulate p53 activation in lung adenocarcinoma (LUAD) cells.MethodsThe endogenous RPL11-binding proteins in A549 cells were pulled down through immunoprecipitation and identified with a proteomics approach. Docking analysis and GST-fusion protein assays were used to analyze the interaction of ribosomal protein S27a (RPS27a) and RPL11. Co-immunoprecipitation and in vitro ubiquitination assays were used to detect the effects of knockdown of RPS27a on the interaction between RPS27a and RPL11, and on p53 accumulation. Cell cycle, apoptosis, cell invasion and migration, cell viability and colony-formation assays were performed in the presence of knockdown of RPS27a. The RPS27a mRNA expression in LUAD was analyzed on the basis of the TCGA dataset, and RPS27a expression was detected through immunohistochemistry in LUAD samples. Finally, RPS27a and p53 expression was analyzed through immunohistochemistry in A549 cell xenografts with knockdown of RPS27a.ResultsRPS27a was identified as a novel RPL11 binding protein. GST pull-down assays revealed that RPS27a directly bound RPL11. Knockdown of RPS27a weakened the interaction between RPS27a and RPL11, but enhanced the binding of RPL11 and murine double minute 2 (MDM2), thereby inhibiting the ubiquitination and degradation of p53 by MDM2. Knockdown of RPS27a stabilized p53 in an RPL11-dependent manner and induced cell viability inhibition, cell cycle arrest and apoptosis in a p53-dependent manner in A549 cells. The expression of RPS27a was upregulated in LUAD and correlated with LUAD progression and poorer prognosis. Overexpression of RPS27a correlated with upregulation of p53, MDM2 and RPL11 in LUAD clinical specimens. Knockdown of RPS27a increased p53 activation, thus, suppressing the formation of A549 cell xenografts in nude mice.ConclusionsRPS27a interacts with RPL11, and RPS27a knockdown enhanced the binding of RPL11 and MDM2, thereby inhibiting MDM2-mediated p53 ubiquitination and degradation; in addition, RPS27a as important roles in LUAD progression and prognosis, and may be a therapeutic target for patients with LUAD.Graphical

Robotic Affinity Purification of Soluble and Insoluble Recombinant Glutathione-S-Transferase Fusion Proteins.

A completely automated purification of glutathione-S-transferase (GST) fusion proteins, either in soluble form or after renaturation of insoluble inclusion bodies, is described. Depending on the expression levels and the amount of glutathione affinity matrix employed, the protocol yields approximately 30-100μg of purified GST-fusion protein from 2mL microplate cultures. The high yield is facilitated by employing an efficient chemical/enzymatic lysis procedure for preparing bacterial cell lysates. Insoluble GST-fusion proteins are automatically refolded by a high-throughput robotic microdialysis procedure that also assesses the degree of successful refolding by integrated GST enzymatic assays and quantitation of soluble protein successfully recovered after affinity purification. For soluble GST-fusion proteins the purification procedure is normally completed within 60min, whereas urea-based denaturation-renaturation strategies typically require an additional 18h. The integration of quantitation of cell growth and affinity-purified GST-fusion protein yield allows direct comparisons of different expression constructs and the yield of soluble GST-fusion proteins to be optimized in a systematic manner.

Development of chimeric receptor activator of nuclear factor-kappa B with glutathione S-transferase in the extracellular domain: Artificial switch in a membrane receptor.

Various chimeric receptors have been developed and used for biological experiments. In the present study, we constructed three types of chimeric receptor activator of nuclear factor-kappa B (RANK) with the glutathione S-transferase (GST) protein in the extracellular domain, and stimulated them using newly synthesized chemical trimerizers with three glutathiones. Although this stimulation did not activate these proteins, we unexpectedly found that the chimera named RANK-GST-SC, in which GST replaced a major part of the RANK extracellular domain, activated nuclear factor-kappa B (NF-κB) signaling approximately sixfold more strongly than wild-type RANK without the ligand. The dimerization of extracellular GST is considered to function as a switch outside the cell, and signal transduction then occurs. GST has been widely employed as a tag for protein purification; GST-fusion protein can be conveniently captured by glutathione-conjugated beads and easily purified from impurity. The present study is a pioneering example of the novel utility of GST and provides information for the development of new chemical biology systems.

SNP rs4971059 predisposes to breast carcinogenesis and chemoresistance via TRIM46-mediated HDAC1 degradation.

Identification of the driving force behind malignant transformation holds the promise to combat the relapse and therapeutic resistance of cancer. We report here that the single nucleotide polymorphism (SNP) rs4971059, one of 65 new breast cancer risk loci identified in a recent genome-wide association study (GWAS), functions as an active enhancer of TRIM46 expression. Recreating the G-to-A polymorphic switch caused by the SNP via CRISPR/Cas9-mediated homologous recombination leads to an overt upregulation of TRIM46. We find that TRIM46 is a ubiquitin ligase that targets histone deacetylase HDAC1 for ubiquitination and degradation and that the TRIM46-HDAC1 axis regulates a panel of genes, including ones critically involved in DNA replication and repair. Consequently, TRIM46 promotes breast cancer cell proliferation and chemoresistance invitro and accelerates tumor growth invivo. Moreover, TRIM46 is frequently overexpressed in breast carcinomas, and its expression is correlated with lower HDAC1 expression, higher histological grades, and worse prognosis of the patients. Together, our study links SNP rs4971059 to replication and to breast carcinogenesis and chemoresistance and support thepursuit of TRIM46 as a potential target for breast cancer intervention.

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Glutathione-Conjugated Hydrogels: Flexible Vehicles for Personalized Treatment of Bacterial Infections.

Skin and soft tissue infections are increasingly prevalent and often complicated by potentially fatal therapeutic hurdles, such as poor drug perfusion and antibiotic resistance. Delivery vehicles capable of versatile loading may improve local bioavailability and minimize systemic toxicities yet such vehicles are not clinically available. Therefore, we aimed to expand upon the use of glutathione-conjugated poly(ethylene glycol) GSH-PEG hydrogels beyond protein delivery and evaluate the ability to deliver traditional therapeutic molecules. PEG and GSH-PEG hydrogels were prepared using ultraviolet light (UV)-polymerization. Hydrogel loading and release of selected drug candidates was examined using UV-visible spectrometry. Therapeutic molecules and GST-fusion protein loading was examined using UV-visible and fluorescent spectrometry. Efficacy of released meropenem was assessed against meropenem-sensitive and -resistant P. aeruginosa in an agar diffusion bioassay. For all tested agents, GSH-PEG hydrogels demonstrated time-dependent loading whereas PEG hydrogels did not. GSH-PEG hydrogels released meropenem over 24h. Co-loading of biologic and traditional therapeutics into a single vehicle was successfully demonstrated. Meropenem-loaded GSH-PEG hydrogels inhibited the growth of meropenem-sensitive and resistant P. aeruginosa isolates. GSH ligands within GSH-PEG hydrogels allow loading and effective delivery of charged therapeutic agents, in addition to biologic therapeutics.

Preparation and application of polyclonal antibody against SSIIb protein from maize

<p id="C3">Antibody is an important reagent for protein function research. It is difficult to carry out protein function research due to the lack of corresponding antibody. Maize starch synthase IIa and starch synthase IIb (SSIIb) belong to starch synthase, which are mainly responsible for the extension of amylopectin. However, the studies of starch synthase IIb were still in lack. In this study, we immunized New Zealand white rabbits with recombinant GST fusion protein (455-704 aa) at the C-terminal of starch synthase IIb as antigen and prepared polyclonal antibody against SSIIb protein. Western blot showed that the polyclonal antibody of SSIIb could recognize not only SSIIb-C (455-704 aa) antigen, but also SSIIb protein in endosperm of maize at different developmental stages. Western blot analysis using the polyclonal antibody revealed that the expression pattern of SSIIb protein in maize endosperm increased first and then decreased from 10 to 30 days after pollination, and the highest expression level was found at 15 days after pollination. Semi-quantitative RT-PCR results indicated that the transcription level of <italic>SSIIb</italic> was the highest at 20 days after pollination. These results indicated that the specific polyclonal antibody against SSIIb was successfully prepared, and it could specifically recognize the endogenous SSIIb protein of maize by Western blot, which laid a foundation for further study of the function of SSIIb protein.

Oxidation of Huntingtin Modulates its Aggregation and Interaction with Membranes

Huntington's disease is an autosomal dominant disease caused by the expansion of a polyglutamine (polyQ) domain within the first exon of the huntingtin (htt) protein. PolyQ expansion promotes htt aggregation into oligomers, b-sheet rich fibrils, and other amorphous species. Htt aggregation is mediated by the first 17 N-terminal amino acids (N17) that directly precede the polyQ domain. In addition, N17 is a membrane binding domain. Both the ability of N17 to enhance htt aggregation and bind lipid membranes are attributed to its ability to form an amphipathic α-helix. N17 also contains several sites for potential posttranslational modifications (PTMs), including oxidation of two methionine residues. Here, the impact of oxidation of methionine residues within N17 on htt aggregation in the presence or absence of lipid membranes comprised of total brain lipid extract (TBLE) was investigated. Two experimental systems were used, a synthetic N-terminal peptide fragment of htt (N17-Q35-P10) and full-length htt-exon1 with 46 repeat glutamines expressed as a GST-fusion protein. For the second system, cleavage of the GST moiety initiates aggregation. To oxidize the methionine residues in N17, the experimental htt systems were treated with hydrogen peroxide. Based on ThT assays, hydrogen peroxide treatment reduced N17-Q35-P10 fibril formation in a dose-dependent manner. Fibrillization of full-length htt-exon1 appeared inhibited with similar hydrogen peroxide treatments as assessed by ex situ atomic force microscopy (AFM) assays. That is, increasing peroxide treatments led to the emergence of shorter and unbranched fibrils while promoting oligomerization. The addition of lipid vesicles generally reduced fibrilization of both experimental systems with and without hydrogen peroxide treatment. However, similar to the trend observed in the absence of lipid, increasing oxidant concentration favored the formation of oligomers. In addition, the aggregation of unoxidized and oxidized htt-exon1 was directly tracked on supported TBLE lipid bilayers using in situ AFM. Both oxidized and unoxidized lipid bilayers were investigated. In general, oxidation of either htt-exon1 alone or both htt and TBLE resulted in reduced membrane damage and disruption. Unoxidized htt deposited quickly and aggressively on TBLE bilayers, forming lumpy amorphous aggregates. With oxidized lipid membranes, htt-exon1 deposited less aggressively on the bilayer, forming a mix of amorphous and smaller grainy oligomeric aggregates over a longer period. In contrast, when both htt-exon1 and TBLE were oxidized, the aggregates formed were mostly grainy oligomeric aggregates. Collectively, these observations suggest that oxidation plays an important role in both the aggregation of htt and its ability to bind lipid membranes, which may ultimately be related to the location of the methionine residues within the amphipathic α-helix of N17.

Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein\u2013protein interactions of the fused protein

Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein–protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST4QN) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST4QN as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein–protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST4QN tag could improve the reproducibility and reliability of GST pulldown experiments.

Detection of antibody to Bacillus anthracis recombinant PA in vaccinated animal serum

Anthrax is a worldwide zoonosis in animals and human. In Mongolia, the confirmed case of anthrax outbreak is reported every year over the past decade. The prevention and control measure of animal anthrax is vaccination using spore of attenuated Sterne strain, but horse does not get vaccinated in Mongolia. In this study, we constructed the recombinant plasmid for over expression of anthrax protective antigen (PA)/GST fusion protein in pGEX-6P-1 vector and purified the recombinant PA (r-PA) using glutathione Sepharose column under native and denaturing conditions. Since both forms of r-PA were recognized by specific antibody against PA, ELISA system to detect antibody titer in vaccinated bovine serum was constructed. Total of 890 vaccinated cattle serum were collected from 178 cattle at 0, 3, 5, 8 and 12 months’ post vaccination. As negative control, 200 cattle serum from Umnugovi aimag were selected which does not have anthrax foci. All serum was tested by rPA indirect ELISA and, antibody to PA were detected in vaccinated cattle serum but were not detected in negative serum. Therefore, rPA should be used in as monitoring of the anthrax vaccination.

Covalent binding of glutathione on magnetic nanoparticles: Application for immobilizing small fragment ubiquitin-like-specific protease 1

Magnetic nanoparticles bound with glutathione (GSH) are useful for diagnostics, enzyme immobilization, and affinity precipitation by using the strong and specific interaction of GSH with glutathione S-transferase (GST)-fused proteins. Our studies revealed that GSH-bound magnetic nanoparticles could be obtained using the covalent bond linkage of GSH and nanoparticles to promote the stability of bound GSH. To yield this conjugate, superparamagnetic iron oxide nanoparticles (SPIONs) were prepared and modified using tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES), which introduced amino groups that were then activated with maleic anhydride (MA) for covalent binding of GSH. After MA was used to activate the amino-grafted SPION for 24 h, the yield of GSH conjugation increased over 4 days from 37 % to 74 % of the original amine density on the surface as the incubation of GSH with MA-activated SPION. These GSH-bound magnetic nanoparticles, designated as SPION@silica-GSH with approximately 103 nmol GSH/mg particles, were ready for coupling with GST-fused protein through the GSH–GST affinity interaction. A GST-tagged small fragment of ubiquitin-like-specific protease 1 (sfULP1) was used as the model protein for immobilization on SPION@silica-GSH. ULP1 is a small ubiquitin-like modifier (SUMO) protease. Results indicated that this immobilized GST-sfULP1 could retain 87 % ± 5 % enzyme activity of free protease before immobilization and could catalyze the cleavage of the SUMO-fused peptide (SUMO-GLP-1) to obtain glucagon-like peptide-1, a peptide hormone for type 2 diabetes therapy.