Part:BBa_K2865001:Design

AR185-T2A-EGFP, nanobody inhibiting RyR2 phosphorylation

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 155
  Illegal NgoMIV site found at 302
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

To obtain antibodies with high affinity to RyR2, we adopt the phage display technique, one of the Nobel Prize technology (Chemistry, 2018). Phage display is a laboratory technique for the study of protein–protein interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In our study, a library of variable domains of camellidae heavy chain-only antibodies (VHH) was constructed. A vast majority of VHH clones were inserted into phagemid and expressed on the surface of the phages. In order to select binders to RyR2, bio-panning was performed with immobilized RyR2 protein. To obtain antibodies that functionally inhibit of RyR2 phosphorylation, antibody fragments isolated in the previous step were tested for its effect in an ELISA based RyR2 phosphorylation assay. Finally, AR185 and a negative control AR117 were obtained for further investigation.

 

Fig.1 Workflow of phage display construction and screening (Díez et al. 2015)

Camel antibody library construction To construct the camel library, peripheral blood mononuclear cells (PBMCs) were isolated from a total of 300ml blood sample. Total mRNA was extracted from PBMCs for cDNA synthesis. VHH genes were cloned by nested PCR from cDNA library as described previously. The final PCR products (~ 400bp) were cloned into the phagemid vector pCANTAB5E and transformed into electro-competent E. coli TG1 cells for the preparation of phages. Phage Display and Biopanning Phages displaying the VHH proteins on its surface were prepared as described previously. For biopanning, we coated 96-well plates with RyR2. The phages were added to each well to allow bingding. After 1 hour of incubation at room temperature, the unbound and nonspecifically bound phages were removed using 5 washes. The specifically bound phage was then eluted and used to infect freshly prepared E. coli TG1 cells. After four rounds of panning, 300 randomly picked clones were analyzed for RyR2 binding by phage ELISA. Among these clones, 276 antibody fragments specifically bound to RyR2. One antibody fragment which did not bind to RyR2 was choose as a negative control, termed as VHH-AR117. ELISA To obtain antibodies that functionally inhibit of RyR2 phosphorylation, each of the antibody fragments was tested for its effect in an ELSA based RyR2 phosphorylation assay. 4 antibody fragments were potent inhibitors of RyR2 phosphorylation. The complementary determining regions (CDRs) were confirmed by sequence analysis and the result revealed that there was only one unique clone in this panel of antibody fragments, termed as VHH-AR185.

 

Figure 2. Isolation of RyR2-specific nanobody by phage display. (A)Phage-displayed nanobody fragments were selected against RyR2 by four rounds of panning. A gradual increase in phage titers was detected after each round of panning. (B) Polyclonal phage ELISA from the output phage of each round of panning. Control group used BSA as the irrelevant antigen. (C) Heat map generated from ELISA data of purified RyR2 channels which were phosphorylated in the presence of the PKA. (D) Kinetic analysis of AR185 binding to RyR2 was performed by SPR.