Literatura académica sobre el tema "FLAG epitope tag"

A FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface. In the screening, SLC35B2, which encodes 3’-phosphoadenosine-5’-phosphosulfate transporter 1, was identified as the candidate gene. The detection of FLAG-tagged misfolded proteins at the cell surface was significantly increased in SLC35B2-knockout cells. Furthermore, protein tyrosine sulfation mediated by tyrosyl-protein sulfotransferase 2 (TPST2) suppressed FLAG-tagged protein detection. Localization analysis of the FLAG-tagged misfolded proteins confirmed that defects in tyrosine sulfation are only responsible for enhancing anti-FLAG staining on the plasma membrane but not inducing the localization change of misfolded proteins on the plasma membrane. These results suggest that a FLAG tag on the misfolded protein would be sulfated, causing a reduced detection by the M2 anti-FLAG antibody. Attention should be required when quantifying the FLAG-tagged proteins in the secretory pathway.

Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals with an almost-worldwide distribution. Conventional FMD vaccines consisting of chemically inactivated viruses have aided in the eradication of FMD from Europe and remain the main tool for control in endemic countries. Although significant steps have been made to improve the quality of vaccines, such as improved methods of antigen concentration and purification, manufacturing processes are technically demanding and expensive. Consequently, there is large variation in the quality of vaccines distributed in FMD-endemic countries compared with those manufactured for emergency use in FMD-free countries. Here, we have used reverse genetics to introduce haemagglutinin (HA) and FLAG tags into the foot-and-mouth disease virus (FMDV) capsid. HA- and FLAG-tagged FMDVs were infectious, with a plaque morphology similar to the non-tagged parental infectious copy virus and the field virus. The tagged viruses utilized integrin-mediated cell entry and retained the tag epitopes over serial passages. In addition, infectious HA- and FLAG-tagged FMDVs were readily purified from small-scale cultures using commercial antibodies. Tagged FMDV offers a feasible alternative to the current methods of vaccine concentration and purification, a potential to develop FMD vaccine conjugates and a unique tool for FMDV research.

ABSTRACT A FLAG epitope tag was substituted within variable loop 1 (V1), 2 (V2), or 4 (V4) of the gp120 envelope glycoprotein of simian immunodeficiency virus strain 239 (SIV239) to evaluate the extent to which each variable loop may serve as a target for antibody-mediated neutralization. Two sites within each variable loop of SIV239 were chosen for individual epitope tag insertions. FLAG epitope substitutions were also made in the V1, V2, and V4 loops of a neutralization-sensitive derivative of SIV239, SIV316. Of the 10 FLAG-tagged recombinant viruses analyzed, three (SIV239FV1b, SIV239FV2b, and SIV239FV4a) replicated with kinetics similar to those of the parental strain, SIV239, in both CEMx174 cells and the immortalized rhesus monkey T-cell line 221. The SIV316FV1b and SIV316FV4a FLAG variants replicated with a substantial lag, and the five remaining recombinants did not replicate detectably. Both gp160 and gp120 from replication-competent FLAG variants could be immunoprecipitated from transfected 293T cells by the anti-gp120 rhesus monoclonal antibody (RhMAb) 3.11H, the anti-FLAG MAb M2, and CD4-immunoglobulin, whereas only unprocessed gp160 was detected in 293T cells transfected with replication-defective variants. Furthermore, gp120 was detectably incorporated only into virions that were infectious. SIV239FV1b was sensitive to neutralization by MAb M2, with a 50% inhibitory concentration of 1 μg/ml. Neither SIV239FV2b nor SIV239FV4a was sensitive to M2 neutralization. The ability of the M2 antibody to neutralize SIV239FV1b infectivity was associated with an increased ability of the M2 antibody to detect native, oligomeric SIV239FV1b envelope protein on the surfaces of cells relative to that for the other SIV FLAG variants. Furthermore, SIV239FV1b was globally more sensitive to antibody-mediated neutralization than was parental SIV239 when these strains were screened with a panel of anti-SIV MAbs of various specificities. These results indicate that the V1 loop can serve as an effective target for neutralization on SIV239FV1b. However, antibody-mediated neutralization of this variant, similar to that of other SIV239 variants that have been studied previously, was associated with a global increase in neutralization sensitivity. These results suggest that the variable loops on the neutralization-resistant SIV239 strain are difficult for antibodies to access effectively and that mutations that allow neutralization have global effects on the trimeric envelope glycoprotein structure and accessibility.

ABSTRACT To understand the molecular determinants of measles virus (MV) cytopathicity, we have characterized mutant viruses exhibiting a more-extensive cell-to-cell fusion while maintaining efficient replication to high titers. A virus which is modified by the addition of an 8-amino-acid Flag epitope tag at the cytoplasmic tail of its H (for MV hemagglutinin) envelope glycoprotein replicates efficiently, has an increased cytopathicity, possesses a greater infectivity per particle, and has an altered protein composition compared with that of unmodified MV. The mutant phenotype is not specifically linked to the epitope sequence, since an alternatively added HA (for influenza virus-derived hemagglutinin) epitope tag caused similar effects. We demonstrate that both epitope tags weaken the interaction between the H and fusion (F) glycoproteins in virus-infected cells. This reduction in strength of H/F interaction is independent of the presence of the viral matrix (M) protein. Viruses with this less stable complex are more sensitive to neutralization by a soluble octameric form of the CD46 receptor, consistent with their increased fusogenicity. Similar analyses of glycoproteins derived from MV strains with reduced cytopathicities confirm that the strength of H and F glycoprotein interaction is a modulator of viral fusogenicity.

Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 μM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571→Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser269) in the cytosol and the analogous residue in ACAT2 (Ser249) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.

We have recently developed surface oligopeptide knock-in for rapid target selection (SORTS), a novel method to isolate mammalian cells with gene modifications using FACS-sorting. It relies on CRISPR/Cas9-mediated targeted knock-in of a very short promoterless expression construct (250 bp) comprising a Flag or HA epitope embedded into the smallest GPI-protein CD52, and a polyA signal from the beta-globin. CD52 efficiently delivers the epitope to the cell surface, where it serves as a marker for selection, while polyA terminates transcription and silences target gene expression. Primarily, SORTS was developed to knock out genes encoding intracellular or secreted proteins, which cannot be used as markers for selection of live cells. Using in-frame modification of SORTS, we demonstrated the possibility of HIV-1 provirus inactivation via sorting of GPI-tag positive cells. In order to make the “cured” cells resistant to a subsequent HIV-1 infection, the epitope tag in the CD52 molecule was substituted by one of the fusion inhibitor peptides from the CHR-domain of gp41. We selected a series of cell-surface-expressed, GPI-anchored, C34-based peptides that confer a strong cellular resistance to HIV-1 infection mediated by NL4-3, JRFL, or ZM153 Env. These findings together with a monoclonal antibody raised against the C34 peptide provide an opportunity to generate and select HIV-resistant lymphocytes for a therapeutic goal. SORTS was also adapted to engineer transgenic HIV-1 effector Т cells and to study cell-to-cell transmission. To facilitate transgenesis, we developed a knock-in strategy to express GPI-tag from the intronic region of the human PPP1R12C gene (AAVS1 locus) and delivered FRT sites of recombination into both alleles. In summary, SORTS is a novel instrument to isolate rare cells with precise genomic modifications with broad applications, including HIV biology. This work was supported by the Russian Science Foundation (grant 18-14-00333) and the Russian Foundation for Basic Research (grants 18-29-07052, 18-04-01016).

Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1). Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.

ABSTRACT Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein+, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.