Methods
Plasmid constructs
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 [8, 9] was cloned in pBUDNeo downstream of the CMV promoter to generate hBirA-GFP pBUDNeo (Fig. 2). In parallel, the N-terminal Avi-TEV-3xFLAG (ATF) and C-terminal 3xFLAG-TEV-Avi (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 hBirA-GFP 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.
Transient transfections
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.
Nuclear extracts
Transiently transfected cells were harvested after 24 h and nuclear extracts were made as previously described [10]. Nuclear proteins were quantitated using Bio-Rad’s colorimetric Protein Assay kit I.
Antibodies
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 Aldrich).
Other methods
Streptavidin pulldown, SDS-PAGE electrophoresis and Western immunoblotting were all done as described in [11]. Streptavidin–horseradish peroxidase (HRP) conjugate was purchased from Perkin Elmer.
Results
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 [12] allowing for the option of tandem affinity purification. The two tags are separated by a TEV protease cleavage site (Fig. 1). The N-terminal Avi-TEV-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 [9].
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 pBUDNeo-based 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 [15, 16], 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 shown).
Discussion
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 [7]). We 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.
