BTag: A novel six-residue epitope tag for surveillance and purification of recombinant proteins

doi:10.1016/0378-1119(95)00795-4

Gene

Volume 169, Issue 1, 22 February 1996, Pages 53-58

https://doi.org/10.1016/0378-1119(95)00795-4 Get rights and content

Abstract

Epitope tagging (Eta) is becoming an increasingly useful technique in molecular biology and biotechnology for the detection, characterisation and purification of recombinant proteins (re-proteins). Here we describe a novel Eta system composed of two different monoclonal antibodies (mAb; D11 and F10) and a 6-amino-acid Eta (Gln-Tyr-Pro-Ala-Leu-Thr or QYPALT). This Eta was derived from a highly conserved region of the major core protein, VP7, of bluetongue (BT) viruses, hence the name BTag. BTag is unique among current tagging systems in its lack of charge and the fact the tag sequence can be placed and detected in any region of a re-protein. Other useful features of BTag include its small size and its recognition by two different mAb. Using the BTag system, more than 30 re-proteins have been produced from a variety of host organisms, and the antigenicity of the tag sequence was maintained in all of the proteins tested to date. Our results demonstrated that BTag could be superior to other existing Eta systems for certain applications

References (14)

D.H. Du Plessis et al.
Fine mapping of a continuous epitope on VP7 of bluetongue virus using overlapping synthetic peptides and a random epitope library
Virology
(1994)
P.A. Kolodziej et al.
Epitope tagging and protein surveillance
Methods Enzymol.
(1991)
R.E. Randall et al.
Two-tag purification of recombinant proteins for the construction of solid matrix-antibody-antigen (SMAA) complexes as vaccines
Vaccine
(1993)
H. Schägger et al.
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa
Anal. Biochem.
(1987)
D.B. Smith et al.
Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase
Gene
(1988)
G.P. Smith et al.
Libraries of peptides and proteins displayed on filamentous phage
Methods Enzymol.
(1993)
L.-F. Wang et al.
Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5
J. Immunol. Methods
(1995)

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Cited by (27)

PhiXing-it, displaying foreign peptides on bacteriophage ΦX174
2016, Virology
Citation Excerpt :
To determine if other insertions were tolerated at Thr21, a variety of peptide encoding DNAs were cloned as described above (Tables 1 and 2). We tested a diverse set of model epitope tags and fusion domains: hemagglutinin (HA) epitope, myc epitope, the Bluetongue virus VP7 epitope tag (BTAG), and the P. Shermanii transcarboxylase domain (PSTCD); as well as bona fide neutralizing epitopes from HPV16 L2 (RG-1), Influenza A virus M2 protein (M2), and the circumsporozoite protein (CS) from Plasmodium falciparum (Gambhira et al., 2007; Murtif et al., 1985; Reddy et al., 2000; Wang et al., 1996; Young et al., 2012; Zavala et al., 1985; Zebedee and Lamb, 1988). These inserts encompass a wide variety of sizes, charges, and side chain chemistries (Table 2).
Although bacteriophage φX174 is easy to propagate and genetically tractable, it is use as a peptide display platform has not been explored. One region within the φX174 major spike protein G tolerated 13 of 16 assayed insertions, ranging from 10 to 75 amino acids. The recombinant proteins were functional and incorporated into infectious virions. In the folded protein, the peptides would be icosahedrally displayed within loops that extend from the protein׳s β-barrel core. The well-honed genetics of φX174 allowed permissive insertions to be quickly identified by the cellular phenotypes associated with cloned gene expression. The cloned genes were easily transferred from plasmids to phage genomes via recombination rescue. Direct ELISA validated several recombinant virions for epitope display. Some insertions conferred a temperature-sensitive (ts) protein folding defect, which was suppressed by global suppressors in protein G, located too far away from the insertion to directly alter peptide display.
Presence of bluetongue virus in the marginal zone of the spleen in acute infected sheep
2011, Veterinary Microbiology
Citation Excerpt :
The tissues were fixed in 10% neutral formalin and embedded in paraffin wax for routine histology, IHC and ISH. A rabbit Anti-B-tag polyclonal antibody (Genscript, Piscataway, NJ, USA), targeted to a six-residue peptide QYPALT on a highly conserved region of VP7 (Wang et al., 1996a,b) was used. The VP7 was selected as the target for both IHC and ISH, since VP7 is the serogroup specificity determinant (Roy and Mertens, 2000) and the most abundant structural protein on BTV virions (Grimes et al., 1998).
Bluetongue virus, a member of the genus Orbivirus of the family Reoviridae, is the causative agent of bluetongue, which is a non-contagious Culicoides mediated blood-borne disease. The present study characterizes the pathogenicity of a Taiwan prototype BTV2/KM/2003 in Corriedale sheep inoculated subcutaneously into the ear pinna. Histologically, multifocal petechiated hemorrhage, with mild to moderate inflammation and edema, were present in the contralateral ear pinna, tongue, and facial skin, without remarkable lesions in lymphoid organs. By days post-infection (DPI) 7, viral VP7 antigen, detected by immunohistochemistry, presented in the spleen, chiefly located in the outer rim of <3 cell thickness of marginal zone macrophages bordering the marginal zone and red pulp, and T lymphocytes of the red pulp. By DPI 11, viral signals shifted from the marginal zone to macrophages and small lymphocytes within follicles of the spleen. In situ hybridization with VP7 gene probe detected strong signals in the spleen, chiefly spanning the whole width of 5–10 cell thickness of the marginal zone, including the marginal zone macrophages and marginal zone B cells, as well as macrophages of sheathed capillaries in the red pulp. This study demonstrates molecular as well as morphologic evidence of the presence of bluetongue virus in the marginal zone of the spleen, most likely associated with viremia in acute infection, as previously demonstrated by the authors.
Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat
2010, Virology
Citation Excerpt :
The genes encoding the p13 and p16 and σB proteins were cloned into two different E. coli expression vectors to enable the production of recombinant fusion proteins with C-terminal epitope tags. The vector prSET-C (Invitrogen) contains a nucleotide sequence encoding a hexahistidine (6 × His) epitope tag adjacent to the multiple cloning site (MCS), and the vector pGD3 is a modified version of pGEX-1N which contains a nucleotide sequence encoding glutathione-S-transferase (GST) adjacent to the MCS (Wang et al., 1996). The prSET-C and the pGD3 vectors were modified to incorporate Asc I and Not I restriction sites and were renamed pHAN and pGAN, respectively.
This report describes the discovery and characterization of a new fusogenic orthoreovirus, Broome virus (BroV), isolated from a little red flying-fox (Pteropus scapulatus). The BroV genome consists of 10 dsRNA segments, each having a 3′ terminal pentanucleotide sequence conserved amongst all members of the genus Orthoreovirus, and a unique 5′ terminal pentanucleotide sequence. The smallest genome segment is bicistronic and encodes two small nonstructural proteins, one of which is a novel fusion associated small transmembrane (FAST) protein responsible for syncytium formation, but no cell attachment protein. The low amino acid sequence identity between BroV proteins and those of other orthoreoviruses (13–50%), combined with phylogenetic analyses of structural and nonstructural proteins provide evidence to support the classification of BroV in a new sixth species group within the genus Orthoreovirus.
Improvement of a recombinant antibody-based serological assay for foot-and-mouth disease virus
2010, Journal of Immunological Methods
Differentiating foot-and-mouth disease virus (FMDV) antibodies generated during a natural infection from those due to vaccination (DIVA) is crucial for proving freedom from disease after an outbreak and allowing resumption of trade in livestock products. The World Organisation for Animal Health (OIE) recommends that FMDV vaccines are composed of inactivated virus that has been purified to remove non-structural viral proteins. Such purified vaccines primarily induce antibodies to viral structural proteins, whereas replicating virus stimulates host antibodies specific for both structural and non-structural proteins. The current preferred FMDV DIVA test is a competitive ELISA (C-ELISA) designed to detect antibodies to the non-structural protein 3ABC. Previously, we described the development of an FMDV DIVA test based entirely on recombinant proteins (the recombinant detecting antibody and the 3ABC coating antigen) produced in Escherichia coli. In this study, we have determined the precise binding site of the recombinant detecting antibody to a conserved sequence within the 3B region of the 3ABC protein, replaced the original E-tag of the detecting antibody with two in-house tags and engineered a direct antibody–reporting enzyme (alkaline phosphatase) fusion protein. These modifications have further improved the DIVA test, providing great potential for large scale production and uptake due to its simplicity, reproducibility and low cost.
Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species
2008, Journal of Immunological Methods
Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS–CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402–622) and the N4 fragment (aa 220–336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS–CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS–CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS–CoV, these assays will be a useful tool to trace the origin and transmission of SARS–CoV and to minimise the risk of animal-to-human transmission.
Genetic analysis of J-virus and Beilong virus using minireplicons
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Citation Excerpt :
The BeiPV L ORF was amplified in four fragments, using the restriction sites PvuII, NheI, StyI, and SalI. The fragments were partially assembled in the pGD3 vector (Wang et al., 1996) before complete assembly in the pTM1 vector. Site directed mutagenesis, as described for the NiV L gene (Magoffin et al., in press), was used to alter the Q residue of the conserved GDNQ sequence into an E residue.
J-virus (JPV), isolated from wild mice in Australia, and Beilong virus (BeiPV), originally isolated from human mesangial cells in China and subsequently detected in rat mesangial cells, represent a new group of paramyxoviruses which have exceptionally large genomes (> 19 kb) and contain more than six transcriptional units. In this study, minireplicons were employed to assess the taxonomic status of JPV and BeiPV. Our results demonstrated that, whilst the genome replication machineries of JPV and BeiPV can be interchanged, they were not functional when exchanged with that of Nipah virus. These studies indicate that JPV and BeiPV are closely related to each other and support the classification of these two viruses into a separate genus. In addition, the minireplicons were also used to demonstrate that these large-genome viruses also comply with the ‘rule of six’ and that over-expression of the C protein has a detrimental effect on minigenome replication.

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Gene

Abstract

References (14)

Fine mapping of a continuous epitope on VP7 of bluetongue virus using overlapping synthetic peptides and a random epitope library

Virology

Epitope tagging and protein surveillance

Methods Enzymol.

Two-tag purification of recombinant proteins for the construction of solid matrix-antibody-antigen (SMAA) complexes as vaccines

Vaccine

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa

Anal. Biochem.

Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase

Gene

Libraries of peptides and proteins displayed on filamentous phage

Methods Enzymol.

Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5

J. Immunol. Methods

Cited by (27)

PhiXing-it, displaying foreign peptides on bacteriophage ΦX174

Presence of bluetongue virus in the marginal zone of the spleen in acute infected sheep

Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat

Improvement of a recombinant antibody-based serological assay for foot-and-mouth disease virus

Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species

Genetic analysis of J-virus and Beilong virus using minireplicons

Short communicationBTag: A novel six-residue epitope tag for surveillance and purification of recombinant proteins

Abstract

Virology

Methods Enzymol.

Vaccine

Anal. Biochem.

Gene

Methods Enzymol.

J. Immunol. Methods

Short communication
BTag: A novel six-residue epitope tag for surveillance and purification of recombinant proteins