IDENTIFICATION OF NEW TAG SEQUENCES WITH DIFFERENTIAL AND SELECTIVE RECOGNITION PROPERTIES FOR THE ANTI-FLAG MONOCLONAL-ANTIBODIES M1, M2 AND M5

The FLAG peptides DYKDDDDK and MDYKDDDDK are widely used affinity tags . Here we describe new variants of the FLAG peptides which, in direct ELISA, showed selective and differential binding to the commercially a vailable anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of th e FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1, M2 and M5. Among the newly identif ied tag sequences are those that bind only one of the anti-FLAG mAbs a nd those that bind only two or all three of the anti-FLAG mAbs. Exampl es of new tag sequences are MDFKDDDDK (which binds mAb M5 and does not bind mAbs Evil and M2) and MDYKAFDNL (which binds mAb M2 and does not bind mAbs M1 and M5). The sensitivity in direct ELISA of some variant s was increased, e.g. using mAb M2 it was found that replacing DDDDK i n MDYKDDDDK by AFDNL increased the sensitivity in ELISA at least 10-fo ld, The activity of this peptide was studied in more detail. In differ ent direct ELISAs, in which MDYKAFDNL was synthesized on polyethylene pins, coated onto polystyrene microtiter plates or onto nitrocellulose paper, the activity of this peptide was similar, i.e. increased at le ast 10-fold over that of MDYKDDDDK. Remarkably, in competitive ELISA t he binding activity of soluble MDYKAFDNL, was decreased 10-fold over t hose of soluble MDYKDDDDK or DYKDDDDK. The results seem to suggest tha t, in solution, the conformation of MDYKAFDNL is more 'unstructured' c ompared to its conformation when coated or linked to a carrier. We pos tulate that the newly described tag sequences may be used as affinity tags to separately detect, quantify and purify multiple co-expressed p roteins and/or subunits.