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.

Keywords: conformation-specific; epitope binning; phage display; recombinant antibodies; “capture and release”.

Fig. 1

Construct design and solubility of CBP-tagged targets. (a) Representation of the expression vector, pCBPH6, used to express CBP-tagged proteins. The vector is an engineered pET28a vector containing T7 promoter. RBS, POI, CBP, Gly-Ser and His₆ stand for ribosome binding site, protein of interest, N5A mutant of calmodulin binding peptide tag (RWKKAFIAVSAANRFKKIS), linker of glycine and serine residues and hexahistidine tag respectively. (b) Solubility comparison of Avi-tagged vs CBP-tagged VENTX. CBP-tagged construct was fairly soluble (red arrow) while there was hardly any protein in soluble fraction from the Avi-tagged construct (blue arrow). U: uninduced cell lysate, I: Induced cell lysate, US: supernatant fraction from uninduced culture, IS: supernatant fraction from induced culture, IP: pellet fraction from induced culture. CBP-tagged target is localized more in IS fraction than Avi-tagged ones. Equal amount of protein was loaded in each well.

Fig. 2

Purification and Characterization of CBP-tagged targets. (a) Analytical size exclusion (aSEC) profile of purified CBP-target (CBP-SETD7) by Ni-NTA chromatography shows a mono dispersed peak lacking any soluble aggregates. (b) Comparison of thermal stability of CBP-tagged proteins (blue) with His₆-tagged (red) and chemically biotinylated (green) protein samples by DSF. Stability is significantly compromised in some cases with chemical biotinylation while no such effect is observed in CBP-tagged constructs.

Fig. 3

CBP-tagged targets are effectively immobilized on CaM coated beads. (a) Pull-down assay: Lane 1: Molecular weight marker (Precision Plus Protein Unstained Standard from Bio-Rad), Lane 2: Input or total protein used (TP), Lane 3: Unbound fraction or flowthrough (FT), Lane 4: Wash (W) fraction, Lane 5: Elution (E) fraction with 10 mM EDTA, Lane 6: Avi-CaM coated SA beads treated with SDS loading buffer. Avi-CaM is highlighted (red asterisk). Almost quantitative capture and elution of the CBP-MBP is observed (red arrow). (b) Recovery of phage displaying MBP binding Fab (MOS1) is maximized (1st panel) only when both CaM and CBP-MBP are used in the mock sorting. The other panels are negative controls where either one of the two or both are missing. SETA1 is phage displaying Fab specific for an unrelated target. Titer values of MOS1 and SETA1 used as inputs were similar.

Fig. 4

Characterization of target-Fab complexes. (a) aSEC profile of individual target (blue) and target-Fab (green) complex. Fabs form high affinity complexes with target proteins that can be separated by SEC. (b) Melting curves of individual targets (green), Fabs (red) and target-Fab (blue) complex. Fabs are thermally very stable (T_m> 70°C) and stabilize the targets in the complex to an appreciable extent (marked by black arrow). Target-Fab complex was prepared by mixing target: Fab in 1:1.5 molar ratio. The melting peak of the excess Fab in the complex at higher temperature is also observed.

Fig. 5

Generation of conformation specific Fabs. (a) Selection strategy to obtain conformation specific Fabs from CBP-tagged targets. CBP-MBP is in open conformation without maltose (green hexagon). In presence of maltose, it adopts the closed conformation. Separate sorting experiments with immobilized targets in specific conformations generate Fabs that are conformation specific. (b) Phage ELISA results show that Fabs generated against closed form of MBP in presence of maltose have a drastically compromised binding to open form of MBP in absence of maltose. (c) SPR sensograms showing that Fabs recognize specific conformation of MBP. Fab 7O binds to open form of MBP immobilized on NTA chip without maltose. No significant binding was observed in 1mM maltose.

Fig. 5

Generation of conformation specific Fabs. (a) Selection strategy to obtain conformation specific Fabs from CBP-tagged targets. CBP-MBP is in open conformation without maltose (green hexagon). In presence of maltose, it adopts the closed conformation. Separate sorting experiments with immobilized targets in specific conformations generate Fabs that are conformation specific. (b) Phage ELISA results show that Fabs generated against closed form of MBP in presence of maltose have a drastically compromised binding to open form of MBP in absence of maltose. (c) SPR sensograms showing that Fabs recognize specific conformation of MBP. Fab 7O binds to open form of MBP immobilized on NTA chip without maltose. No significant binding was observed in 1mM maltose.

Fig. 6

Generation of epitope specific Fabs. (a) “Epitope exclusion” sorting strategy to generate Fabs binding to epitope other than the immunodominant one (shown in red arrow). The antigen was pre-incubated with excess of Fab23 so that the immunodominant epitope is masked, driving the phage pool to bind to other epitopes during selection. Excess of Fab23 in the solution blocks the epitope and also helps to wash away the binders specific for the masking Fab. (b) Competitive phage ELISA results show that Fabs generated by “epitope masking” bind to SETD7 even in presence of 1uM Fab23 (green bars) but successfully competed off by 50nM SETD7 (blue bars). This proves that these Fabs are very specific and bind to epitopes different from Fab23 which are available even in presence of 1 uM Fab23. (c) Epitope binning experiment by protein ELISA proves that the Fabs bind to an epitope distinct from Fab23 as they are not competed by Fab23. Fab1E, Fab3E, Fab4E and Fab6E compete among themselves for the same epitope. (d) Epitope binning experiment using SPR. His₁₀-SETD7 is immobilized on NTA sensor chip. (1) A mixture of Fab4E + Fab23 (blue sensogram) is injected after saturating the ligand surface with first analyte (Fab23). 2^nd injection with Fab23 alone is used as control (red sensogram). Overlay of the two sensograms shows a net increase in signal intensity when Fab4E + Fab23 is injected indicating that Fab4E is binding to an epitope that is non overlapping with the epitope to which Fab23 binds. (2) Same pattern is observed on reversing the order of analyte injection. Here the ligand is first saturated with Fab4E followed by injecting a mixture of Fab23 +Fab4E (red sensogram). Control experiment was done using Fab4E alone in the second injection (blue sensogram). Similar increase in net signal intensity in comparison to control further confirms sequential binding of the Fabs to different epitopes on SETD7.

Fig. 6

Generation of epitope specific Fabs. (a) “Epitope exclusion” sorting strategy to generate Fabs binding to epitope other than the immunodominant one (shown in red arrow). The antigen was pre-incubated with excess of Fab23 so that the immunodominant epitope is masked, driving the phage pool to bind to other epitopes during selection. Excess of Fab23 in the solution blocks the epitope and also helps to wash away the binders specific for the masking Fab. (b) Competitive phage ELISA results show that Fabs generated by “epitope masking” bind to SETD7 even in presence of 1uM Fab23 (green bars) but successfully competed off by 50nM SETD7 (blue bars). This proves that these Fabs are very specific and bind to epitopes different from Fab23 which are available even in presence of 1 uM Fab23. (c) Epitope binning experiment by protein ELISA proves that the Fabs bind to an epitope distinct from Fab23 as they are not competed by Fab23. Fab1E, Fab3E, Fab4E and Fab6E compete among themselves for the same epitope. (d) Epitope binning experiment using SPR. His₁₀-SETD7 is immobilized on NTA sensor chip. (1) A mixture of Fab4E + Fab23 (blue sensogram) is injected after saturating the ligand surface with first analyte (Fab23). 2^nd injection with Fab23 alone is used as control (red sensogram). Overlay of the two sensograms shows a net increase in signal intensity when Fab4E + Fab23 is injected indicating that Fab4E is binding to an epitope that is non overlapping with the epitope to which Fab23 binds. (2) Same pattern is observed on reversing the order of analyte injection. Here the ligand is first saturated with Fab4E followed by injecting a mixture of Fab23 +Fab4E (red sensogram). Control experiment was done using Fab4E alone in the second injection (blue sensogram). Similar increase in net signal intensity in comparison to control further confirms sequential binding of the Fabs to different epitopes on SETD7.

Fig. 7

Generation of domain specific Fabs. (a) “Domain exclusion” sorting strategy to generate Fabs exclusively to N-terminal domain (yellow). The C-terminal domain (red) is used in huge excess as soluble competitor to enrich for binders specific for N-terminal domain. Phages binding to the C-terminal domain are washed away. (b) Competitive phage ELISA results show that Fabs are not competed by C-terminal domain of SETD7 (blue bars) but by full length construct of SETD7 (green bars). This proves that the sABs bind to N- terminal domain of SETD7.

Fig. 8

Immunoprecipitation (IP) and immunofluorescence (IF). (a) Native IP: Western Blot analysis showing antiSETD7 Fabs are able to recognize and pull down overexpressed Flag-tagged SETD7 in native form from HEK293 cells. The protein is entirely in the flowthrough (FT) (lane 2) when the cell lysate is incubated with empty beads. No band is seen in the bead fraction (B) (lane 3). Similar effect is observed with an unrelated anti-GFP Fab (H3) coated beads. The protein is observed in flowthrough (lane 4) and no band in bead fraction (lane 5). Beads coated with anti SETD7 Fabs (3E, 4E, 5E and 6E) capture Flag-tagged SETD7 in native form almost quantitatively from HEK293 lysate. (b) Immunofluorescence: Immunostainings of MEFs overexpressing wild type SETD7 (wtSETD7) with Fab3E as a primary antibody and secondary antihuman Alexa-488 conjugated IgG. Nuclear localization of wtSETD7 is observed. Hardly any signal was obtained with the negative controls −/− MEFs that proves the specificity of Fab3E.