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Specific antibody interactions with short peptides have made epitope tagging systems a vital tool employed in virtually all fields of biological research. Here, we present a novel epitope tagging system comprised of a monoclonal antibody named GD-26, which recognises the TD peptide (GTGATPADD) derived from Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant. The crystal structure of the antigen-binding fragment (Fab) of GD-26 complexed with the TD peptide was determined to a resolution of 1.45 A. The TD peptide was found to adopt a 310 helix conformation within the binding cleft, providing a characteristic peptide structure for recognition by GD-26 Fab. Based on the structure information, polar and non-polar forces collectively contribute to the strong binding. Attempts to engineer the TD peptide show that the proline residue is crucial for the formation of the 310 helix in order to fit into the binding cleft. Isothermal calorimetry (ITC) reported a dissociation constant KD of 12 ± 2.8 nM, indicating a strong interaction between the TD peptide and GD-26 Fab. High specificity of GD-26 IgG to the TD peptide was demonstrated by Western blotting, enzyme-linked immunosorbent assays (ELISA) and immunofluorescence as only TD-tagged proteins were detected, suggesting the effectiveness of the GD-26/TD peptide tagging system. In addition to already-existing epitope tags such as the FLAG tag and the ALFA tag adopting either extended or α-helix conformations, the unique 310 helix conformation of the TD peptide together with the corresponding monoclonal antibody GD-26 offers a novel tagging option for research.

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here, we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron-transfer high-energy collision dissociation (EThcD), on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.

Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein’s function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin’s central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin’s known receptors, calculating the KD of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.

As an indispensable structure protein, the herpes simplex virus 1 (HSV-1) UL6 has been described to exert numerous roles in viral proliferation. However, its exact subcellular localization and subcellular transport mechanism is not well known. In the present study, by utilizing confocal fluorescent microscopy, UL6 was shown to mainly locate in the nucleus in enhanced yellow fluorescent protein or Flag tag fused expression plasmid-transfected cells or HSV-1-infected cells, whereas its predicted nuclear localization signal was nonfunctional. In addition, by exploiting dominant negative mutant and inhibitor of different nuclear import receptors, as well as co-immunoprecipitation and RNA interference assays, UL6 was established to interact with importin α1, importin α7 and transportin-1 to mediate its nuclear translocation under the help of Ran-mediated GTP hydrolysis. Accordingly, these results will advance the knowledge of UL6-mediated biological significances in HSV-1 infection cycle.

Detecting protein-protein interactions (PPIs) is one of the most used approaches to reveal the molecular regulation of protein of interests (POIs). Immunoprecipitation of POIs followed by mass spectrometry or western blot analysis enables us to detect co-precipitated POI-binding proteins. However, some binding proteins are lost during cell lysis or immunoprecipitation if the protein binding affinity is weak. Crosslinking POI and its binding proteins stabilizes the PPI and increases the chance of detecting the interacting proteins. Here, we introduce the method of DSP (dithiobis(succinimidyl propionate))-mediated crosslinking, followed by tandem immunoprecipitation (FLAG and HA tags). The eluted proteins interacting with POI can be analyzed by mass spectrometry or western blotting. This method has the potential to be applied to various cytoplasmic proteins. Graphical abstract.

The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.

The shelterin protein complex protects natural chromosome ends from being recognized as DNA damage sites and also regulates the synthesis of telomeric repeats by telomerase. TPP1, a shelterin subunit that is essential for telomerase extension of telomeres, has been studied intensively in recent years. Many such studies utilize epitope tagged TPP1, but it is unclear how the tags may affect the multiple cellular functions of TPP1. Here we analyzed the effect of adding a 3x Flag epitope tag to the N- or C-terminus of TPP1. While the position of the tag did not affect TPP1's interaction within the shelterin complex or its localization to telomeres, the N-terminal Flag tag on TPP1 impaired telomerase function, resulting in reduced telomerase processivity in vitro and a failure to stimulate telomere elongation in vivo. The C-terminally Flag-tagged TPP1, in contrast, behaved similarly to untagged TPP1 in all functional aspects examined. These findings suggest that caution is required when utilizing epitope tagged TPP1 to study its regulation of telomerase function.

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-021-01210-7.

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.

Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.

Protein fusion tags are important tools in research when robust methods for protein purification and detection are required. In this chapter we present an efficient method for stringent protein purification. A small domain, denoted ABDz1, with affinity for both human serum albumin and Protein A has been developed. The purification tag is based on an albumin-binding domain from Streptococcal Protein G that was engineered to bind Protein A. The ABDz1-tag can be fused to any protein of choice and the purification can be performed using standard laboratory equipment. In this chapter a method for purification of ABDz1-tagged proteins using two successive affinity purification steps is described.

Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of protein interaction networks. In instances where a suitable antibody is not available, it is common to graft synthetic tags onto a target protein sequence thereby allowing the use of commercially available antibodies for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the selective elution of protein complexes, competitive displacement using a large molar excess of the tag peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis can be applied to FLAG-Tag co-immunoprecipitation experiments to increase the depth of protein coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without the need for additional sample handling or altering sample preparation protocols. SIGNIFICANCE: We have shown that application of FAIMS separation in the gas phase can increase the proteome coverage of Flag-Tagged co-immunoprecipitation mass spectrometry experiments versus one without FAIMS. We were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without additional sample handling, fractionation, machine run time or modifying the sample preparation protocol.

Altered serotonin (5-HT) levels contribute to disease states such as depression and anxiety. Synaptic serotonin concentration is partially regulated by the serotonin transporter (SERT), making this transporter an important therapeutic target. This study seeks to examine the lipid accessible domains of hSERT to provide critical information regarding the apo-state of this transporter in a lipid environment. Recombinant hSERT was inducibly expressed in a human cell line. Solubilized SERT was purified by affinity chromatography using a FLAG Tag and reconstituted into mixed lipid vesicles containing our photoactivatable lipid probe. The lipid-accessible domains of the reconstituted transporter in membranes in its apo-state were probed via photocrosslinking to azi-cholesterol followed by quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LC-MS). MS studies identified crosslinks in three transmembrane loops consistent with the known topology of SERT. Surprisingly, the amino- and carboxy-terminal domains were similarly crosslinked by cholesterol, suggesting that these regions may also be intimately associated with the lipid bilayer. The data presented herein assist in further refining our understanding of the topography of the apo-state of hSERT via analysis of lipid accessibility.

Recombinant proteins have increased our knowledge regarding the physiological role of proteins; however, affinity purification tags are often not cleaved prior to analysis, and their effects on protein structure, stability and assembly are often overlooked. In this study, the stabilizing effects of an N-terminus dual-FLAG (FT(2)) tag fusion to transthyretin (TTR), a construct used in previous studies, are investigated using native ion mobility-mass spectrometry (IM-MS). A combination of collision-induced unfolding and variable-temperature electrospray ionization is used to compare gas- and solution-phase stabilities of FT(2)-TTR to wild-type and C-terminal tagged TTR. Despite an increased stability of both gas- and solution-phase FT(2)-TTR, thermal degradation of FT(2)-TTR was observed at elevated temperatures, viz., backbone cleavage occurring between Lys9 and Cys10. This cleavage reaction is consistent with previously reported metalloprotease activity of TTR [Liz et al. 2009] and is suppressed by either metal chelation or excess zinc. This study brings to the fore the effect of affinity tag stabilization of TTR and emphasizes unprecedented detail afforded by native IM-MS to assess structural discrepancies of recombinant proteins from their wild-type counterparts.

The S-adenosylmethionine carrier (SAMC) is a membrane transport protein located on the inner membrane of mitochondria that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix. SAMC mutations can cause a series of mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation and biosynthesis. Here, we describe the expression, purification and oligomerization of SAMC. The SAMC genes from three species were cloned into a eukaryotic expression vector with a GFP tag, and confocal microscopy analysis showed that these SAMCs were localized to mitochondria. A BacMam expression system was used for the expression of D. rerio SAMC with a FLAG tag. A size-exclusion chromatography analysis showed that SAMC may form a hexamer. A negative-staining electron microscopy analysis showed that SAMC formed tiny uniform particles and also confirmed the oligomerization of SAMC.

FLAG tag (DYKDDDDK) is a short peptide commonly used for the purification of recombinant proteins. The high price of the affinity columns and their limited reusability are a shortcoming for their widespread use in biotechnology applications. Molecularly imprinted polymers (MIPs) can circumvent some of the limitations of bioaffinity columns for such applications, including long-term stability, reusability, and cost. We report herein the synthesis of MIPs selective to the FLAG tag by hierarchical imprinting. Using the epitope imprinting approach, a 5-amino acid peptide DYKDC was selected as a template and was covalently immobilized on the surface of microporous silica beads, previously functionalized with different aminosilanes, namely, 3-(2-aminoethylamino)propyldimethoxymethylsilane, AEAPMS, and N-(2-aminoethyl)-2,2,4-trimethyl-1-aza-2-silacyclopentane, AETAZS. We investigated the effect of the type of silane on the production of homogeneous silane-grafted layers with the highest extent of silanol condensation as possible using 29Si CP/MAS NMR. We observed that the right orientation of the imprinted cavities can substantially improve analyte recoveries from the MIP. After template and silica removal, the DYKDC-MIPs were used as sorbents for solid-phase extraction (molecularly imprinted solid-phase extraction) of the FLAG peptide, showing that the polymer prepared with AETAZS-bound silica beads contained binding sites more selective to the tag (RMIP-AZA = 87.4% vs RNIP-AZA = 4.1%, n = 3, RSD ≤ 4.2%) than those prepared using AEAPMS (RMIP-DM = 73.4% vs RNIP-DM = 23.2%, n = 3, RSD ≤ 4.0%) as a functionalization agent. An extensive computational molecular modeling study was also conducted, shedding some light on the interaction mechanism between the FLAG peptide and the imprinted template in the binding cavities., This work was supported by the Spanish Ministry of Economy and Competitiveness (grant RTI2018-096410-B-C21). Wethank Prof. M. J. Torralvo for assistance with the N2adsorption experiments and the National Centre for Electron Microscopy and the Centre for Elemental Microanalysis(UCM, Madrid) for the technical support. N.G. thanks theSpanish Ministry of Economy and Competitiveness(MAT2016-81001-P project) and Dr. LevíLópez (Character-ization Service of ICTP-CSIC) for performing solid-stateNMR spectroscopy. Y.R. thanks Dr. Mihaly Mezei (Depart-ment of Pharmacological Sciences, Icahn School of Medicineat Mount Sinai) for critically reading the computational part ofthis manuscript and suggesting improvements. Computationswere supported in part through the computational resourcesand staffexpertise provided by the Scientific ComputingFacility at the Icahn School of Medicine at Mount Sinai.

It has been shown that the single-domain intrabody 2H9-L against the hepatitis C virus (HCV) capsid (core) protein inhibits the viral propagation and NF-κB promoter activity induced by the HCV core. In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1–123 amino acid residues (C123), 1–152 amino acid residues (C152), 1–177 amino acid residues (C177) and 1–191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E. coli and then purified. Approximately 175 and 132 μg of the intrabody were purified from 100 ml of E. coli culture and 10 silkworm pupae, respectively, by affinity chromatography. The C123, C152, C177 and C191 HCV core protein variants were purified to approximately 152, 127, 103 and 155 μg, respectively, from 100 ml of E. coli culture. An ELISA in which the intrabodies were immobilized revealed that the intrabodies purified from both hosts were bound to all HCV core protein variants. However, their binding to the C191 appeared to be weak compared to their bindings to the other HCV core protein variants. When C152 was immobilized in the ELISA, the binding of each intrabody to the core protein was also observed. These purified intrabodies can be used in biochemical analyses of the inhibitory mechanism of HCV propagation and as protein interference reagents, thus providing a potential pathway to developing a new type of antiviral drug.

The budding yeast and model eukaryote Saccharomyces cerevisiae has been invaluable for purification and analysis of numerous evolutionarily conserved proteins and multisubunit complexes that cannot be readily reconstituted in Escherichia coli. For many studies, it is desirable to functionalize a particular protein or subunit of a complex with a ligand, fluorophore or other small molecule. Enzyme-catalysed site-specific modification of proteins bearing short peptide tags is a powerful strategy to overcome the limitations associated with traditional nonselective labelling chemistries. Towards this end, we developed a suite of template plasmids for C-terminal tagging with short peptide sequences that can be site-specifically functionalized with high efficiency and selectivity. We have also combined these sequences with the FLAG tag as a handle for purification or immunological detection of the modified protein. We demonstrate the utility of these plasmids by site-specifically labelling the 28-subunit core particle subcomplex of the 26S proteasome with the small molecule fluorophore Cy5. The full set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).

The G-H loop of the foot-and-mouth disease virus (FMDV) virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid (RGD) motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious cDNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker (DYKDDDDK) was inserted upstream (–4) or downstream (+10) of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAG tag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites (RGD–4 and RGD+10) tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals (DIVA).

Οbjective To construct mammalian expression vectors for the N- or C-terminal tagging of proteins with a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag. Results We constructed and tested by transient transfections mammalian expression vectors for the co-expression from a single plasmid of N- or C-terminally tagged proteins bearing a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag separated by a tobacco etch virus (TEV) protease cleavage site, together with a mammalian codon-optimized BirA biotin ligase fused to green fluorescent protein. We also describe platform vectors for the N- or C-terminal AVI-TEV-FLAG tagging of any complementary DNA of choice. These vectors offer versatility and efficiency in the application of metabolic biotinylation tandem affinity tagging of nuclear proteins in mammalian cells. Electronic supplementary material The online version of this article (10.1186/s13104-018-3500-9) contains supplementary material, which is available to authorized users.

Recombinant viruses of strain Ingelvac® PRRS porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus vaccine were produced with two individual small in-frame deletions in nonstructural protein 2 (nsp2; Δ23 and Δ87) and also the same deletions supplanted with foreign tags (Δ23-V5, Δ23-FLAG, Δ23-S, Δ87-V5, Δ87-FLAG, Δ87-S). The viruses, but one (Δ87-FLAG), were stable for 10 passages and showed minimal effects on in vitro growth. Northern hybridization showed that the Δ23-tagged probe detected intracellular viral genome RNA as well as shorter RNAs that may represent heteroclite species, while the Δ87-tagged probe detected predominantly only genome length RNAs. When the tagged viruses were used to probe nsp2 protein in infected cells, perinuclear localization similar to native nsp2 was seen. Dual infection of Δ23-S and Δ87-S viruses allowed some discrimination of individual tagged nsp2 protein, facilitating future research. The mutants could potentially also be used to differentiate infected from vaccinated animals.

Many viruses often have closely related yet antigenically distinct serotypes. An ideal vaccine against viral infections should induce a multivalent and protective immune response against all serotypes. Inspired by bacterial membrane vesicles (MVs) that carry different protein components, we constructed an agr locus deletion mutant of the Staphylococcus aureus strain (RN4220-Δagr) to reduce potential toxicity. Nanoscale vesicles derived from this strain (ΔagrMVs) carry at least four major components that can deliver heterologous antigens. These components were each fused with a triple FLAG tag, and the tagged proteins could be incorporated into the ΔagrMVs. The presentation levels were (3.43 ± 0.73)%, (5.07 ± 0.82)%, (2.64 ± 0.61)%, and (2.89 ± 0.74)% of the total ΔagrMV proteins for Mntc-FLAG, PdhB-FLAG, PdhA-FLAG, and Eno-FLAG, respectively. With two DENV envelope E domain III proteins (EDIIIconA and EDIIIconB) as models, the DENV EDIIIconA and EDIIIconB delivered by two staphylococcal components were stably embedded in the ΔagrMVs. Administration of such engineered ΔagrMVs in mice induced antibodies against all four DENV serotypes. Sera from immunized mice protected Vero cells and suckling mice from a lethal challenge of DENV-2. This study will open up new insights into the preparation of multivalent nanosized viral vaccines against viral infections.

In this study, purification of recombinant human growth hormone (rhGH) with FLAG tag from E. coli inclusion bodies was described. The step-by-step process carried out was as follows: bacterial cells were suspended in lysis buffer and disrupted in a single sonication step. Then, inclusion bodies were isolated and solubilized in different buffers to obtain the best one which was found to be Tris buffer containing 2% deoxycholate with a satisfyingly adequate capacity to dissolve inclusion bodies at pH 12.5. In the third step, the proteins in solubilizing buffer were refolded by being diluted in refolding buffer. This was carried out with lowering the pH value to 8 using a direct dilution (to five volumes) process. Following a specific enterokinase cleavage for removing the tag, the rhGH was purified by ion-exchange chromatography. Following with the research, for the first time, a comparative study was performed on two weak ion-exchangers, namely DEAE and CM-Sepharose (two most commonly used resins), so as to investigate their pH dependence and resolution. DEAE-Sepharose at pH 8.25 exhibited the best overall performance and efficiency for rhGH purification as measured by either yield or purity. The overall process was reproducible and easy to scale up, making it a suitable choice for large-scale production of therapeutic proteins.

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity, and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization - denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) - are revisited with focus on the protein, and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design., This study was funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal, under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01- 0145-FEDER-006684) and the Post-Doctoral grant SFRH/BPD/ 110640/2015, and by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) supported by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l-1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris.

Detection, purification and characterization of proteins are essential procedures in the field of biochemistry. Epitope tag systems are commonly used to characterize unknown proteins. The Deinococcus radiodurans bacterial phytochrome (DrBphP) protein has been used as an antigen to generate anti-DrBphP mouse monoclonal antibodies and to identify their specific epitopes. Among these antibodies, the 2B8 monoclonal antibody recognizes an epitope of 9 amino acids (RDPLPFFPP). The 2B8 epitope does not match the amino acid sequence for any known protein. On Western blot analysis, the 2B8 antibody showed strong and highly specific interactions with the 2B8 epitope. These results suggest that the 2B8 epitope-antibody is a useful epitope tag system for protein characterization. In addition, we generated a modified epitope (RDPLPAFPP) via point mutation in a previous study. This modified epitope showed significantly increased reactivity with the 2B8 antibody. In this study, we developed a protein purification system using the 2B8 epitope tag and antibody. 2B8 antibodies were bound to protein G-agarose beads as affinity ligands. Recombinant DrBphP proteins were then exposed to 2B8 antibody-bound protein G-agarose beads. Bound DrBphP proteins were then eluted by competition with the original or modified 2B8 epitope peptides. DrBphP proteins were successfully purified via an affinity chromatography system using a 2B8 original peptide and even better purified using the 2B8 modified peptide. These findings indicate that the 2B8 epitope tag system is a better tool for protein detection and purification.

Rapid and high-throughput protein purification methods are required to explore structure and function of several uncharacterized proteins. Isolation of recombinant protein expressed in Escherichia coli strain BL21 (DE3) depends largely on the efficient and speedy bacterial cell lysis, which is considered as the bottleneck during protein purification. Cells are usually lysed by either sonication or high pressure homogenization, both of which are slow, require special equipment, lead to heat generation, and may result in loss of protein's biological activity. We report here a novel method to lyse E. coli, which is rapid, and results in high yield of isolated protein. Here, we have carried out intracellular expression of lysozyme domain (LD) of mycobacteriophage D29 endolysin. LD remains non-toxic until chloroform is added into the culture medium that permeabilizes bacterial cell membrane and allows the diffusion of LD to the peptidoglycan layer causing latter's degradation ensuing cell lysis. Our method efficiently lyses E. coli in short duration. As a proof-of-concept, we demonstrate large scale isolation and purification of α subunit of E. coli RNA polymerase and GFP, when they are co-expressed with LD. We believe that our method will be adopted easily in high-throughput as well as large scale protein isolation experiments.

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography.

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.

Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.

Ribosomal S6 protein kinase 4 (RSK4) is an important novel tumor suppressor that inhibits breast cancer cell growth and induces senescence. However, RSK4m, which is a variant of RSK4 resulting from alternative splicing, may play distinct roles in some aspects. Knowledge about the mechanisms of RSK4 or RSK4m activity has been lacking. Analysis of the RSK4 and RSK4m interactome could provide insight into their specific functions and integrative mechanisms. Using tandem affinity purification, we obtained protein complexes that interacted with RSK4 or RSK4m. Mass spectrum analysis was performed to identify the obtained protein complexes, and bioinformatics analysis was performed. In this study, we isolated and identified 82 RSK4-associated proteins and 137 RSK4m-associated proteins using two STREP II and a single Flag tag-based tandem affinity purification (SF-TAP) coupled with nano LC-MS/MS in MDA-MB-231 cells. Gene Ontology and Ingenuity Pathway Analysis analyses provided functional annotations and protein-protein interaction networks of the protein interactome of RSK4 and RSK4m. Functional annotations of these proteins by bioinformatics analyses highlight the essential role of RSK4 and RSK4m in coordination with their interacting partners to mediate multiple biological processes, especially cell senescence. Moreover, after comparing the interactome of RSK4 and RSK4m, we found that RSK4m is involved in more molecular functions than RSK4.

Protein purification typically involves expressing a recombinant gene comprising a target protein fused to a suitable affinity tag. After purification, it is often desirable to remove the affinity tag to prevent interference with downstream functions of the target protein. This is mainly accomplished by placing a protease site between the tag and the target protein. Typically, a small oligopeptide ‘stub’ C-terminal to the cleavage site remains attached to the target protein due to the requirements of sequence-specific proteases. Furthermore, steric hindrance can also limit protease efficiency. Here, we show that respectively fusing the interacting ePDZ-b/ARVCF protein-peptide pair to the target protein and a protease enables efficient processing of a minimised sequence comprising only residues N-terminal to the cleavage site. Interaction of the protein-peptide pair enforces proximity of the protease and its minimised cleavage sequence, enhancing both catalysis of a sub-optimal site and overcoming steric hindrance. This facilitates the high yield purification of fully native target proteins without recourse to specialised purification columns. NMRC (Natl Medical Research Council, S’pore) Published version

Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-γ (His-BoIFN-γ) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 × 10(4) units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-γ (rBoIFN-γ) was purified to 98.3% purity with 63% recovery. The rBoIFN-γ had an antiviral activity of 1.6 × 10(3) units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification.