Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase
Ioannou et al. BMC Res Notes
Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase
Marina Ioannou 0
Dimitris N. Papageorgiou 2 3
Vasily Ogryzko 1
John Strouboulis 0
0 Institute of Molecular Biology and Biotechnology, Foundation of Research & Technology Hellas , 100 Nikolaou Plastira Street, 70013 Heraklion, Crete , Greece
1 UMR8126, Université Paris-Sud 11, CNRS, Institut de Cancérologie Gustave Roussy , 94805 Villejuif , France
2 Division of Molecular Oncology, Biomedical Sciences Research Center “Alexander Fleming” , 34 Fleming Street, 166 72 Vari , Greece
3 Present Address: Division of Proteomics Stem Cells and Cancer, German Cancer Research Center (DKFZ) , Im Neuenheimer Feld 581, 69120 Heidelberg , Germany
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.
Expression vectors; Tandem affinity purification; Metabolic biotinylation tagging; Avi tag; FLAG tag; BirA biotin ligase
Metabolic biotinylation tagging of proteins offers a high
affinity tagging approach with an increasing number
of applications in mammalian cells [
]. It involves the
co-expression in cells of the E. coli BirA biotin ligase
together with the protein of interest fused to a small
artificial peptide tag (the Avi tag) which is specifically
recognized and efficiently biotinylated by BirA in cells [
Biotin-tagged proteins can be bound very tightly by
avidin and streptavidin (dissociation constant Kd = 10−15), a
fact that has been widely exploited in many affinity-based
biochemical applications . Furthermore, biotinylation
tagging offers a number of advantages for the purposes
of protein tagging. First, there are only five, mostly
mitochondrial, naturally biotinylated proteins ensuring low
nonspecific background [
]. Second, high stringencies
can be employed in any biotin/(strept)avidin affinity
purification or detection protocol, without fear of losing the
tagged protein. Third, a great variety of biotin/(strept)
avidin-related reagents are commercially available for
protein applications. Lastly, protein biotin tagging can be
further extended by combining combination with other
epitope tags, fused in tandem to the protein of interest
However, as versatile as biotinylation tagging may be,
it is somewhat complicated by the fact that it is a binary
system relying on the simultaneous expression of the
Avi-tagged protein of interest and of the BirA protein
biotin ligase. In addition, expression of the prokaryotic
BirA biotin ligase in mammalian cells can be problematic
due to inefficient translation as a result of differences in
codon usage between bacterial and mammalian cells [
In order to overcome these challenges, we describe here
the construction of mammalian expression vectors for
the expression of tagged proteins bearing an N- or
C-terminal Avi-triple FLAG tandem affinity tag and,
concurrently, of a mammalian codon-optimized (“humanized”)
BirA-GFP fusion. The N- or C-terminally tagged protein
and hBirA-GFP are driven by two separate promoters on
the same plasmid and can be used for transient or stable
transfections in mammalian cells.
Expression vectors were constructed using the
mammalian expression vector pBudCE4.1 (Life Technologies)
modified by the addition of the thymidine
kinase-neomycin resistance gene (TK NeoR) gene to yield vector
pBUDNeo. The mammalian codon-optimized
(“humanized”) hBirA-GFP fusion [
] was cloned in pBUDNeo
downstream of the CMV promoter to generate
hBirAGFP pBUDNeo (Fig. 2). In parallel, the N-terminal
Avi-TEV-3xFLAG (ATF) and C-terminal
3xFLAG-TEVAvi (FTA) tandem affinity tag sequences (Fig. 1) were
assembled by gene synthesis (GeneArt, Life
Technologies), verified by sequencing and cloned in pBluescript
SK (Agilent Technologies) (for ATF, Additional file 1:
Figure S1A) or pBluescript KS (Agilent Technologies)
(for FTA, Additional file 1: Figure S1B) to generate two
general-purpose plasmids carrying the N- or C-terminal
tandem affinity tagging sequences. Next, the N-terminal
ATF or C-terminal FTA tagging sequences were cloned
downstream of the EF1α promoter in plasmid
hBirAGFP pBUDNeo, to generate plasmids N-ATF/hBirA or
C-FTA/hBirA (Fig. 2a). The GATA1 expression
constructs were generated by in-frame cloning of the GATA1
cDNA to the N-terminal ATF/hBirA vector or the
C-terminal FTA/hBirA vector. The GATA-1 fusions to the tags
in the final expression plasmids were verified by
sequencing. Further details regarding the construction of the
plasmids described here are available upon request.
HEK293 cells (60–70% confluency) were transiently
transfected using the JetPEI™ DNA transfection reagent
according to the manufacturer’s instructions (Source
Bioscience, Nottingham, UK). 8–10 μg of plasmid DNA was
used per 10 cm plate transfected.
Transiently transfected cells were harvested after 24 h
and nuclear extracts were made as previously described
]. Nuclear proteins were quantitated using Bio-Rad’s
colorimetric Protein Assay kit I.
Anti-GATA-1 N6 rat monoclonal antibody (sc-265, Santa
Cruz Biotechnology); anti-GFP a mouse monoclonal
antibody (sc-9996, Santa Cruz Biotechnology); anti-HA
rabbit polyclonal antibody (sc-805, Santa Cruz
Biotechnology); M2 FLAG mouse monoclonal antibody (Sigma
Streptavidin pulldown, SDS-PAGE electrophoresis and
Western immunoblotting were all done as described in
]. Streptavidin–horseradish peroxidase (HRP)
conjugate was purchased from Perkin Elmer.
We generated a series of constructs for the N- or
C-terminal biotinylation tagging of proteins which include
a triple (3x) FLAG tag fused in tandem to the Avi
biotinylatable tag [
] allowing for the option of tandem
affinity purification. The two tags are separated by a
TEV protease cleavage site (Fig. 1). The N-terminal
AviTEV-3xFLAG and the C-terminal 3xFLAG-TEV-Avi
sequences were first cloned in pBluescript SK and KS,
respectively (Additional file 1: Figure S1), thus
generating two platform constructs that can be used for cloning
any cDNA of interest in-frame to the N- or C-terminal
tags, followed by re-cloning of the tagged sequences to an
expression vector of choice or to a gene locus of interest,
for example by CRISPR/Cas9 mediated approaches.
With the aim of generating a single construct for the
expression of either the N-terminally or C-terminally
tagged nuclear protein of interest and of the mammalian
codon optimized hBirA biotin ligase, we used the
pBudNeo expression vector which contains two independent
transcription units driven by the elongation factor 1α
(EF1α) and cytomegalovirus (CMV) promoters (Fig. 2)
and which is well suited for stable or transient
mammalian cell transfections. The N- or C-terminal tandem
affinity tags were cloned under the control of the EF1α
promoter using restriction sites that allow the in-frame
cloning of cDNAs by PCR, whereas the hBirA-GFP
fusion was cloned under the control of the CMV
promoter (Fig. 2a, b). The hBirA biotin ligase-GFP fusion
allows one to use GFP fluorescence to assess transfection
efficiency and hBirA expression levels and to sort
transfected cells from a pool of cells [
In order to test these constructs, we cloned the murine
GATA1 cDNA in-frame to the N- or C-terminal tags
downstream of the EF1α promoter and transiently
transfected them in HEK293 cells. GATA1 is an
essential hematopoietic transcription factor which has been
studied extensively through the application of
biotinylation tagging [
11, 13, 14
]. Nuclear extracts were isolated
at 24 h post-transfection and expression of hBirA-GFP
was confirmed using an anti-GFP antibody (Fig. 3a). We
next confirmed expression of N- or C-terminally tagged
GATA1, as detected by anti-GATA1 and anti-FLAG
antibodies, whereas biotinylation of tagged GATA1 was
confirmed using streptavidin–HRP (Fig. 3a). We also
tested the efficiency of biotinylation mediated by the
mammalian codon optimized hBirA compared to the
original bacterial BirA biotin ligase. To this end, we
transiently transfected HEK293 cells with the
pBUDNeobased vector expressing the C-terminally tagged GATA1
together with hBirA-GFP (Fig. 2b) or with an identical
vector expressing the E. coli 3xHA-tagged BirA instead
of hBirA-GFP. We used dilutions of nuclear extracts
normalized for GATA1 expression from the two
transfections (with hBirA or E. coli BirA) to assay for
biotinylation of tagged GATA1. From this it is clear that hBirA
is more efficient in biotinylating tagged GATA1, since
stronger signals using streptavidin–HRP were obtained
throughout the hBirA nuclear extract dilutions compared
to the BirA dilutions (Fig. 3b).
We also used streptavidin pulldowns in order to assess
the biotinylation efficiency of the N- or C-terminally tagged
GATA1 protein. In both cases, as detected by GATA1
antibody and streptavidin–HRP, we saw that almost all of the
biotin-tagged GATA1 protein is bound and pulled down
by streptavidin beads, indicating a very high efficiency
of tagged GATA1 biotinylation in HEK293 cells (Fig. 3c).
In addition, we also show that streptavidin pulldown of
N-terminally or C-terminally tagged GATA1 results in
the co-precipitation of the endogenous transcription
factor ZNF143 which has been previously reported to
interact with GATA1 [
], thus demonstrating the utility of
these constructs in investigating protein–protein
interactions. Similar results were also obtained in
immunoprecipitation experiments using an anti-FLAG antibody (data not
We describe here the generation of expression vectors for
the efficient biotinylation tagging of proteins in mammalian
cells. Specifically, we generated two platform constructs
bearing in tandem 3xFLAG and biotinylatable Avi tags for
the N- or C-terminal tagging of target proteins of
interest, which can then be re-cloned into mammalian
expression vectors of choice. The presence of two affinity tags in
tandem and of an intervening TEV protease cleavage site
allows downstream tandem affinity purification of tagged
proteins from nuclear extracts (for example, see [
also generated mammalian expression vectors carrying on
the same plasmid N- or C-terminal 3xFLAG and Avi
tandem affinity tags under the control of the EF1α promoter
and the mammalian codon optimized hBirA fused to GFP
under the control of the CMV promoter. These vectors
allow for transient or stable expression and biotinylation
in mammalian cells of N- or C-terminally tagged proteins
using a single plasmid as vector. All the above vectors
provide utility and flexibility in affinity purification protocols
employing in vivo metabolic biotinylation tagging and the
advantages associated with it.
The expression vectors described here rely on their
transient or stable transfection in cultured mammalian cells.
As such, they are subject to the limitations of transfection
assays such as low transfection efficiencies and low levels,
or altogether absent, expression as a result of
chromosomal position effects at the site of integration in stably
transfected cells. Furthermore, expression levels of cDNAs
cloned in the expression vectors described here cannot be
in any way adjusted, as for example in inducible expression
systems. This may result in situations where overexpression
of a given cDNA cloned in the expression vectors described
here may prove deleterious to the cells.
Additional file 1: Figure S1. Restriction maps of plasmid
Avi-TEV3xFLAG_pBS SK (A) and of plasmid 3xFLAG-TEV-Avi_pBS KS (B).
TEV: tobacco etch virus; GFP: green fluorescent protein; cDNA:
complementary DNA; Kd: dissociation constant; TK: thymidine kinase; NeoR: neomycin
resistance; HRP: horseradish peroxidase; EF1α: elongation factor 1α; CMV:
MI and DP carried out experiments and co-authored the manuscript; VO
provided essential reagents for the experimental work; JS conceived the present
study, designed experiments and co-authored the manuscript. All authors
read and approved the final manuscript.
None to declare.
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or
analysed during the current study. All reagents generated in the present study
are freely available to the research community.
Consent to publish
Ethics approval and consent to participate
Work described here was supported in part by National Institutes of Health
(NIH) Grant RO1DK083389 to J.S.
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published maps and institutional affiliations.
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