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Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5

Content Provider	Springer Nature Link
Author	Slootstra, J. W. Kuperus, D. Plückthun, A. Meloen, R. H.
Copyright Year	1997
Abstract	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 available anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of the FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1, M2 and M5. Among the newly identified tag sequences are those that bind only one of the anti-FLAG mAbs and those that bind only two or all three of the anti-FLAG mAbs. Examples of new tag sequences are MDFKDDDDK (which binds mAb M5 and does not bind mAbs M1 and M2) and MDYKAFDNL (which binds mAb M2 and does not bind mAbs M1 and M5). The sensitivity in direct ELISA of some variants was increased, e.g. using mAb M2 it was found that replacing DDDDK in MDYKDDDDK by AFDNL increased the sensitivity in ELISA at least 10-fold. The activity of this peptide was studied in more detail. In different 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 least 10-fold over that of MDYKDDDDK. Remarkably, in competitive ELISA the binding activity of soluble MDYKAFDNL was decreased 10-fold over those of soluble MDYKDDDDK or DYKDDDDK. The results seem to suggest that, in solution, the conformation of MDYKAFDNL is more ‘unstructured’ compared to its conformation when coated or linked to a carrier. We postulate that the newly described tag sequences may be used as affinity tags to separately detect, quantify and purify multiple co-expressed proteins and/or subunits.
Starting Page	156
Ending Page	164
Page Count	9
File Format	PDF
ISSN	13811991
Journal	Molecular Diversity
Volume Number	2
Issue Number	3
e-ISSN	1573501X
Language	English
Publisher	Kluwer Academic Publishers
Publisher Date	1997-01-01
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Affinity tag FLAG peptide Differential recognition Analytical Chemistry Organic Chemistry Polymer Sciences Pharmacy
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Medicine Drug Discovery Molecular Biology Physical and Theoretical Chemistry Information Systems Catalysis Inorganic Chemistry

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