Epitope tag-antibody combination useful for the detection of protein expression in prokaryotic and eukaryotic cells.

Epitope tagging has proven a useful method for monitoring recombinant proteins expressed in either prokaryotic or eukaryotic cells. For this purpose, the epitope should not be present on any of the host cell’s proteins, it should not interfere with either structure or function of the tagged protein, and the corresponding antibody must be of high specificity and affinity. To this day, only a few epitope-antibody combinations have been found satisfactory (5). We describe the successful application of a new epitope-antibody combination. The epitope consists of a 10amino acid sequence motif present in profilin from birch pollen. It is recognized by the monoclonal anti-birch profilin antibody 4A6, which has been characterized as an immunoglobulin IgG2a (1). Epitope recognition by 4A6 is restricted to profilins of the birch tree family (4) and has been demonstrated in immunoblotting, immunoprecipitation and immunofluorescence (1,3). A DNA cassette coding for the 4A6 epitope (S F P Q F K P Q E I) (4) with an EcoRI-compatible 5′ end and an XbaI-compatible 3′ overhang was generated by annealing the synthetic oligonucleotides BiPro1 (5′-AAT TCC TTC CCA CAG TTT AAG CCT CAG GAA AT-3′) and BiPro2 (5′-CTA GAT TTC CTG AGG CTT AAA CTG TGG GAA GG-3′). The resulting doublestranded DNA fragment was cloned into the EcoRI/XbaI-digested vectors pMal/c (New England Biolabs, Schwalbach, Germany) and pcDNA3 (Invitrogen, Leek, Netherlands) to produce a prokaryotic and a eukaryotic expression vector, respectively. The resulting constructs pMal/BiPro and pcDNA3/BiPro were verified by DNA sequencing. Using the C-terminal birch profilin tag, one can monitor: (i) the expression in transformed E. coli of any protein as a fusion protein with the maltose-binding protein (MBP); (ii) its purity after amylose affinity chromatography; and (iii) its integrity after cleaving the N-terminal MBP by factor Xa (according to the instructions by the manufacturer). We tested the usefulness of pMal/ BiPro in this context by cloning into it the cDNA of mouse adenylyl cyclaseassociated protein (CAP; GenBank Accession No. L12367). The 1.4-kb DNA fragment was fused in-frame to the MBP and the epitope tag as an EcoRI fragment. The resulting construct (pMal-CAPBiPro) was verified by restriction analysis and DNA sequencing. Expression of the tagged fusion protein was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and monitored by immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE; Figure 1A). The tagged MBPCAP fusion protein (99 kDa) was faithfully and specifically recognized by 4A6 (Figure 1A, lane 2′). To test the eukaryotic expression vector pcDNA3/BiPro, we inserted a sequence encoding amino acids 858– 1066 of chicken vinculin (GenBank Accession No. J04126) as a HindIII/ EcoRI fragment into the HindIII/ EcoRI-digested pcDNA3/BiPro to generate pcDNA3-VTBiPro. The expression of the tagged polypeptide was monitored in a mammalian epithelial cell line (PtK2) and transfected by the CaPO4 precipitation method. Immunoblot analysis with 4A6 proved the