title: A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.
abstract: Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD~2 pM), which can be reversed by EDTA allowing controlled "capture and release" of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. KD values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Somnath
authors.initials: S
authors.lastname: Mukherjee
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Marcin
authors.initials: M
authors.lastname: Ura
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Hoey
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA; Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA. Electronic address: koss@bsd.uchicago.edu.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D002460:Cell Line; D061505:Cell Surface Display Techniques / Q000379:methods*; D057809:HEK293 Cells; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D011485:Protein Binding / Q000235:genetics; D011496:Protein Methyltransferases / Q000235:genetics; D020703:ras GTPase-Activating Proteins / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D058619:Maltose-Binding Proteins; C505592:NEDD9 protein, mouse; D020703:ras GTPase-Activating Proteins; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; C562044:Setd7 protein, mouse
keywords: conformation-specific; epitope binning; phage display; recombinant antibodies; “capture and release”