Glutathione S-transferase Tag Research Articles

Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.

Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.

Fluorescein-labeled ThUBD probe for super-sensitive visualization of polyubiquitination signal in situ cells

Ubiquitin-binding domains (UBDs) are modular elements that bind non-covalently to the ubiquitin and ubiquitin chains. The preferences of UBDs for ubiquitin chains of specific length and linkage are central to their functions. We demonstrated that an artificial tandem hybrid UBD (ThUBD) exhibits an unbiased high affinity to all ubiquitin chains and is a promising tool for global ubiquitination profiling research. In this study, we labeled fluorescein on the four cysteine residues in the N-terminal glutathione S-transferase (GST) tag of ThUBD, generating a fluorescein-labeled ThUBD (ThUBD-Flu) probe for direct polyubiquitination signal imaging and visualization. Compared to the canonical ubiquitin antibody method, the ThUBD-Flu is hyper-sensitive and accurate to detect ubiquitination signal. More importantly, the ThUBD-Flu probe provided, for the first time, a widely applicable, super-sensitive and unbiased technique for in situ detection of intracellular polyubiquitination signal through immunofluorescence staining, which was only achievable with recombinant fluorescence tag fused ubiquitin gene previously. We propose that ThUBD-Flu, combined with evolving microscopy technology, could serve as prototypes to track and trace cellular polyubiquitination signal in vivo.

Generic Plug-and-Play Strategy for High-Throughput Analysis of PTM-Mediated Protein Complexes.

Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.

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.

Hemalin from Haemaphysalis flava ticks: cloning, expression and antithrombogenicity.

Hemalin, initially described in Haemaphysalis longicornis, is a protein with anticoagulant activity. We retrieved a gene fragment functionally annotated as hemalin from H. flava salivary gland transcriptomic library, but its full-length complementary DNA (cDNA) and antithrombogenicity have not been investigated in the species. Here we cloned the full length of hemalin (Hf-hemalin) by 3'-end rapid-amplification of cDNA ends, and the open reading frame (ORF) of Hf-hemalin was expressed in Escherichia coli. The recombinant protein (rHf-Hemalin) was tested for antithrombogenicity. The full-length of Hf-hemalin was 607 bp with an ORF of423 bp. Protein encoded by Hf-hemalin was predicted to contain 2 Kunitz domains and a signal peptide. The expression of Hf-hemalin in salivary glands, midguts and ovaries was higher in the semi-engorged than the fully engorged. Prokaryotic expression yielded a product of 40 kDa containing a glutathione S-transferase (GST) tag. Incubation of rHf-Hemalin with rat plasma significantly extended prothrombin time and activated partial thromboplastin time compared with normal saline and GST controls. Our data demonstrated that Hemalin from H. flava shared a similar primary structure with that from H. longicornis, and was also anticoagulant. Further investigations are needed to test its feasibility to be an antigen candidate for the development of vaccines against ticks.

Cloning, Expression, and Immunological Characterization of Formosan Subterranean Termite (Blattodea: Rhinotermitidae) Arginine Kinase.

Several parts of the world regularly consume termites. Arthropod arginine kinase proteins often cross-react with human immunoblobulin E (IgE) antibodies and they are considered pan-allergens. The Formosan subterranean termite Coptotermes formosanus (C. formosanus (Shiraki) [Isoptera: Rhinotermitidae]), along with cockroaches, belong to the order Blattodea and they are common household pests in tropical and subtropical parts of the world. An sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band migrating at approximately 37 kDa in C. formosanus termite extracts cross-reacted with IgE from five cockroach allergic patient samples by immunoblot. Liquid chromatography-mass spectrometry analysis of gel slices from the corresponding region of a gel indicated several peptides from the excised region were identical to the American cockroach arginine kinase allergen, Per a 9. The sequence of the full-length C. formosanus arginine kinase gene indicates the protein it encodes is 96% identical to American cockroach Per a 9, 94% identical to German cockroach Bla g 9, and 82-84% identical to shrimp arginine kinase proteins Pen m 2, Lit v 2, and Cra c 2. Full-length C. formosanus arginine kinase was fused to a glutathione S-transferase tag and recombinantly expressed and purified from Escherichia coli by affinity chromatography. The recombinant protein was recognized by IgE from 11 of 12 cockroach or shrimp allergic samples, but did not cross-react with dust mite allergic or peanut/tree nut allergic samples. The results of this study indicate the C. formosanus arginine kinase cross-reacts with cockroach and shrimp allergic IgE, and if consumed would likely act as an allergen.

Purification and activity of the first recombinant enzyme for biodegrading hepatotoxin by Sphingopyxis sp. USTB-05

Cyanobacterial hepatotoxins which are produced and released by bloom-forming cyanobacteria may threaten the safety of drinking water for human beings and animals. Microcystinase (MlrA) catalyzes the first and most important step for biodegrading hepatotoxic microcystins (MCs) and nodularin (NOD), which hydrolyzes the cyclic hepatotoxin into the linear hepatotoxin as the first product. Here the recombinant MlrA of Sphingopyxis sp. USTB-05 for biodegrading hepatotoxins was firstly purified and its activity was also investigated. The purification process of MlrA was comprised of four steps, affinity purification of glutathione S-transferase (GST)-tagged MlrA, thrombin cleavage of GST tag, GST tag removal and thrombin removal with the benzamidine sepharose. The purified recombinant MlrA was found to have a strong ability to catalyze hepatotoxins, and initial microcystin-LR (MC-LR) of 19.9 mg/L and NOD of 25.2 mg/L were removed within 10 min and 12 h at the protein concentration of 60 mg/L, respectively. The results indicated that the enzyme MlrA with a high activity could be purified and obtained with the biotechnology process of expression and purification described here, which is very important in further studies on the enzyme structure and the enzymatic mechanism for biodegrading hepatotoxins.

Construction of E. coli-Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria.

Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexa-histidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.

Expression and purification of recombinant Lactobacillus casei bacteriocin and analysis of its antibacterial activity

ABSTRACT Diseases caused by foodborne pathogens have largely promoted the search for natural biopreservatives. We constructed recombinant plasmids pGEX-4 T-1-lacA, pGEX-4 T-1-lacB, and pGEX-4 T-1-lacAB, harboring genes for bacteriocins lactocin (Lac) A, B, and AB, respectively, with protein expression achieved in Escherichia coli BL21 cells. LacAB was purified using a glutathione S-transferase (GST) protein purification kit, and the GST tag was removed using thrombin for a maximum concentration yield of 1.85 mg/mL. LacAB and the combination of LacA and LacB showed better antimicrobial activity than LacA or LacB alone. The antibacterial activity assay indicated that purified LacAB has broad-spectrum and significant antimicrobial activity against Staphylococcus aureus ATCC25923, with a minimum inhibitory concentration of 125 μg/mL. This study is the first, to our knowledge, to demonstrate the antimicrobial activity of LacAB; moreover, it provides an efficient strategy for the production of bacteriocin LacAB, which could be a food preservative candidate.

Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array

BackgroundThe identification and characterization of epitopes facilitate the discovery and development of new therapeutics, vaccines and diagnostics for infectious diseases. In this study, we developed a glutathione S-transferase (GST)-peptide fusion protein microplate array for the identification of linear B-cell epitopes and applied this novel method to the identification of linear B-cell epitopes of SjSP-13, an immunodiagnostic biomarker of schistosomiasis japonica.MethodsSjSP-13 was divided into 17 overlapped peptides (p1-17), and the coding sequence of each peptide was obtained by annealing two complementary oligonucleotides. SjSP-13 peptides were expressed by fusion with an N-terminal GST tag and a C-terminal 6xHis tag. The GST-peptide-His fusion protein was specifically bound to the Immobilizer Glutathione MicroWell 96-well plates without purification. SjSP-13 peptides and core epitopes that could be recognized by sera from schistosomiasis patients were identified by ELISA and confirmed by Western blot analysis. The receiver operating characteristic (ROC) analysis was performed to determine the diagnostic validity of the identified peptide.ResultsFull-length GST-peptide-His fusion proteins were successfully expressed and specifically bound to the Immobilizer Glutathione MicroWell 96-well plates. Two adjacent peptides (p7 and p8) were found to be highly immunogenic in humans. The core epitope of p7 and p8 is an 11-aa peptide (80KCLDVTDNLPE90) and an 8-aa peptide (90EKIIQFAE97), respectively. The area under the ROC curve (AUC) value of the peptide which contains the two identified epitopes is 0.947 ± 0.019. The diagnostic sensitivity and specificity of the peptide is 76.7% (95% CI: 68.8–84.5%) and 100%, respectively.Conclusions90EKIIQFAE97 and 80KCLDVTDNLPE90 are the two linear epitopes of SjSP-13 recognized by patient sera, and could be potential serological markers for schistosomiasis japonica.

Mapping a highly conserved linear neutralizing epitope at the N-terminus of the gD glycoprotein of bovine herpesvirus type I using a monoclonal antibody

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae subfamily, causes significant economic losses to the cattle industry worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an essential role in the viral entry into permissive cells and possibly cooperates with other envelope glycoproteins. The herpesvirus gD induces a protective immune response against diseases in cattle or animal models. Mapping epitopes on gD will facilitate the understanding of the BoHV-1 pathogenesis and development of alternative vaccines against various diseases associated with the virus. In this study, a monoclonal antibody (MAb), designated as 3C1, was generated using naive BoHV-1 in vaccination of mice, demonstrating that 3C1 was specific to gD and represents a neutralizing activity against BoHV-1 infection in Madin–Darby bovine kidney cells. Panels of overlapping gD recombinant proteins with glutathione S-transferase tag were prepared to define the epitope recognized by 3C1. The data demonstrated that the N-terminus of gD 23APRVTVYVD31 was recognized by 3C1. Furthermore, the 26VTVYVD31 motif was the minimal amino acid sequence for the recognition. The epitope identified in this study is highly conserved among the typical strains of BoHV-1 and BoHV-5, suggesting that this epitope may be useful in the diagnosis of diseases. In addition, the defined region on gD of BoHV-1 might be essential in viral entry upon comparison with the prototype virus in herpes simplex virus (Alphaherpesvirinae). The data will elucidate the roles of gD of BoHV-1 in viral entry and pathogenesis and its potential application for the development of vaccine candidates and diagnostic techniques based on the conserved epitopes on gD or in combination with those of other herpesvirus glycoproteins.

In vivo and in vitro characterization of hydrophilic protein tag-fused Ralstonia eutropha polyhydroxyalkanoate synthase

Polyhydroxyalkanoates (PHAs) are synthesized by bacteria as an intracellular storage polyester, where PHA synthase (PhaC) catalyzes the polymerization of its substrate hydroxyacyl-coenzyme A (HA-CoA) to form PHA. When PhaC is overexpressed in Escherichia coli, most PhaC protein is produced as insoluble inclusion bodies due to its low aqueous solubility. This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaCRe) by fusing a hydrophilic tag, glutathione S-transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaCRe. However, an in vitro assay revealed that the surface of GST-fused PhaCRe (GST-PhaCRe) had increased hydrophilicity, and tended to form correct PhaCRe dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaCRe displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaCRe showed higher catalytic activity than PhaCRe in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaCRe.

Isolation and characterization of soluble human full-length TDP-43 associated with neurodegeneration.

The involvement of transactivation response (TAR) DNA-binding protein 43 (TDP-43) in neurodegenerative diseases was revealed in 2006, when it was first reported to be the main component of the intracellular inclusions in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. After 12 yr it is not yet possible to purify to a reasonable yield and in a reproducible manner a stable full-length protein, which has limited so far the characterization of its structure, function, molecular interactors, and pathobiology. Using a novel protocol we have achieved the purification of the full-length TDP-43, with both a short pectate lyase B tag and a glutathione S-transferase tag, which consisted in its expression in bacteria, solubilization from inclusion bodies, purification under denaturing conditions, refolding, and a final size exclusion chromatography (SEC) step. Differential scanning fluorimetry was used to find the best buffers and combination of additives to increase both its solubility and its stability. The protein is pure, as determined with electrophoresis, Western blotting, and mass spectrometry; properly refolded, as revealed by circular dichroism and fluorescence spectroscopies; functional, because it binds to DNA and protein partners; and stable to degradation and aggregation in a physiologic solution. Analyses with dynamic light scattering and SEC revealed that the protein is a dimer.-Vivoli Vega, M., Nigro, A., Luti, S., Capitini, C., Fani, G., Gonnelli, L., Boscaro, F., Chiti, F. Isolation and characterization of soluble human full-length TDP-43 associated with neurodegeneration.

Expression, purification and characterization of diguanylate cyclase from Rhodococcus ruber

Diguanylate cyclases (DGCs) were responsible for the synthesis of second messenger cyclic di-guanosine monophosphate (c-di-GMP), which were involved in various physiological activities of bacterial species. Here, a full-length DGC from Rhodococcus ruber SD3 fused with glutathione-S-transferase (GST) was expressed in E. coli and purified by glutathione agarose resin. The apparent molecular mass of one subunit of the purified diguanylate cyclase with GST tag (GST-DGC) was estimated to be 71.9 kDa by SDS-PAGE, which was approximately in accordance with the theoretical value of 73.0 kDa. The sequence of GST-DGC was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The blue native PAGE indicated that GST-DGC formed octamer. The optimum pH and temperature for GST-DGC activity were 8.0 and 47 °C, respectively. The fusion protein exhibited high thermostability, and 94% of activity was retained when the protein was incubated at 87 °C for 1 h. Moreover, the fusion protein showed pH stability. The Km, Vmax and Kcat values for GST-DGC enzyme were 9.8 μM, 0.7 μM/min and 1.3 S-1. Some ions such as Zn2+, Mn2+, Fe2+, Ni2+ and Co2+ had inhibitory effects on the activity of the protein, while other ions such as Mg2+, K+ and Na+ slightly activated the protein. The fusion protein also showed rather high stability in the presence of toluene, cyclohexane and n-hexane.

Enhanced recombinant insulin production in transgenic Escherichia coli that heterologously expresses carrot heat shock protein 70

Abstract Recombinant insulin production was enhanced in transgenic Escherichia coli (E. coli BL21, DE3) that heterologously expressed carrot (Daucus carota L.) heat shock protein 70 (DcHsp70). The gene encoding DcHsp70 with the constitutive bacterial lipoprotein promoter was inserted into the E. coli genome using lambda Red-mediated homologous recombination. Nucleotide sequences for the human proinsulin, insulin B and insulin A chains were separately cloned into the pVFT 2S expression vector containing a 6xhistidine and glutathione S-transferase (GST) tag. When induced by isopropyl β- d -1-thiogalactopyranoside treatment, the expression levels of the proinsulin and insulin A and B chain fusion proteins were higher in the transgenic cell lines that heterologously expressed DcHsp70 than in the control cell line. The 6xHis-GST tag was removed from the three versions of the fusion proteins by tobacco etch virus protease. As an active molecular chaperone, DcHsp70 could increase recombinant insulin production when heterologously expressed in E. coli. Our results suggest a possible use of plant Hsps as effective molecular chaperones to increase recombinant protein production in E. coli.

A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism

Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN proto-oncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAM binding primes SYK for rapid LYN-mediated phosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival.

Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector.

An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5'-CCGG-3' of the duplex DNA (ds-DNA) were methylated by M. SssI CpG methyltransferase (M. SssI MTase), the symmetrical sequences 5'-CCGG-3' in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5'-CCGG-3' sequences were specifically cleaved by HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out atpH7.4in the presence of 50μM thionine and 0.5mM H2O2. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. SssI MTase can be determined in the activity range of 0.5-125 unit·mL-1 with a detection limit of 0.2 unit·mL-1 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489μA·μM-1·cm-2. Graphical abstract Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use ofglutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).

Mapping a highly conserved linear neutralizing epitope on gD glycoprotein of bovine herpesvirus type I using a monoclonal antibody.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae, causes a variety of diseases, which result in significant economic losses worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an important role in viral entry into the permissive cells, and protective immune response. The fine mapping epitope on the gD will contribute to the understanding of viral pathogenesis and development of alternative vaccines against various diseases associated with BoHV-1. We previously reported the preparation of a monoclonal antibody (MAb) 2B6, which was raised by a truncated recombinant gD protein, demonstrating a neutralizing activity against BoHV-1 infection in Madin–Darby bovine kidney cells. This study described the identification of a linear B-cell epitope on gD using MAb 2B6. A series of partially overlapping gD proteins with glutathione S-transferase tag were generated to define the epitope recognized by MAb 2B6. The amino acid (aa) sequence 323GEPKPGPSPDADRPE337 was recognized by MAb 2B6 using Western blot with the variedly truncated recombinant proteins. Importantly, this epitope was highly conserved among the typical members of BoHV-1, indicating that the epitope may be utilized in diagnosis of diseases due to BoHV-1 infection. Furthermore, the minimal linear epitope sequence 323GEPKPGP329 on gD recognized by MAb 2B6 was confirmed using single-aa residue deletion mutation in carboxyl terminal. This finding not only contributes to our understanding of gD of BoHV-1 virion but also shows a potential for the development of vaccine candidates and diagnostic techniques.

Enhancement of a protocol purifying T1 lipase through molecular approach

T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.

Identification of an alternative translation initiation site in the sequence of the commonly used Glutathione S-Transferase tag

A large number of proteins are expressed in fusion with a tag to perform their purification. Glutathione-S-Transferase (GST) is a widely used tag to achieve passenger protein purification. Accordingly, commonly used commercial expression vectors contain the coding sequence of GST in order to express fusion proteins. However, fusion proteins are sometimes expressed in a truncated form that may result from an incomplete synthesis, proteolytic cleavage or the presence of a functional alternative translation initiation site. In particular, a truncated as well as a full-length fusion protein were observed when expressing RGS9-1 Anchor Protein without its C-terminal segment (bR9AP) in fusion with GST. Moreover, this truncated protein was found to be purified together with the full-length fusion protein. Here, we identified for the first time an alternative translation initiation site within the sequence of GST that likely becomes accessible for translation only when it is fused with a passenger protein. Indeed, bioinformatics analyses suggest that the secondary structure of the mRNA of the GST-bR9AP fusion protein is different from that of GST alone, which likely allows accessibility of an alternative Shine-Dalgarno sequence coupled with an additional initiation codon within the sequence of GST. The functionality of this alternative translation initiation site was confirmed by site-directed mutagenesis, which resulted in the absence of a truncated fusion protein and, consequently, only a purified full-length fusion protein. This is an extremely important finding in view of the wide use of GST as a purification and solubility-enhancing tag.