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 ~ 2pM), 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 of the Fabs were in sub 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, so the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of 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.

Phage Display Derived Monoclonal Antibodies: From Bench to Bedside

“…Although biopanning of immobilized antigen on solid surfaces is robust, it is often limited by the availability of purified protein and the possibility of its altered conformation when attached to solid surfaces. Therefore, alternative methods, such as in-solution biopanning, followed by affinity capture of antigens tagged with biotin ( 64 ), or calmodulin binding peptide have been used ( 65 ).…”

Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

“…After four rounds of selection, clones with high binding affinity and good competition ratio were chosen based on single-point competitive phage ELISA and sequenced to identify unique binders. The unique binders were cloned in vector pRH2.2, expressed in E.coli BL21(gold) cells and purified following published protocols 34 .…”

The structure of the C-terminal domain of the nucleoprotein from the Bundibugyo strain of the Ebola virus in complex with a pan-specific synthetic Fab

“…The initial screening to identify high-affinity binders was performed using a single-point competitive phage ELISA procedure (Paduch et al, 2013;Mukherjee et al, 2015). This procedure is high-throughput, allowing many potential clones to be evaluated for each target.…”