Ligand regulated protein-protein interaction system
A protein interaction and ligand technology, applied in the field of ligand-regulated protein-protein interaction systems, can solve problems such as undesired heterodimerization
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Embodiment 1
[0125] Embodiment 1: LRPPI system based on RBP4
[0126] For bovine and human RBP4, a series of ligands are known to induce conformational changes in the loop region, which lead to dissociation of the native protein partner TTR (see prior art cited above). In this example, it is demonstrated by using human RBP4 and its synthetic non-retinoid ligand A1120 (PubChem CID 25138295) how this ligand-induced conformational switch of lipocalin-folded molecules can be used as components. To this end, His-tagged full-length RBP4 (UniProt ID P02753) was used as an antigen in an alternate screening process in the presence (5 μM) and absence of A1120, in which the ligand-dependent lipocalin fold bound Interaction partners were selected from a library of two scaffolds with different protein structures, namely FN3 and Sso7d-based scaffolds (Traxlmayr et al., J Biol Chem. 2016; 291(43):22496-22508; Chen et al., Methods Enzymol .2013;523:303-326). As described below, this approach yields a l...
Embodiment 2
[0142] Example 2: Identification of clinically applicable lipocalin folding ligands with the ability to induce conformational transitions in lipocalin folding
[0143] By using human RBP4 and its natural interacting partner TTR, we exemplify a strategy for the identification of novel lipocalin folding ligands with the ability to induce lipocalin folding conformational switches. To identify clinically applicable lipocalin fold ligands, we performed several databases (World Drug Index, KEGG Medicus , KEGGLigands, DrugBank, Human Metabolome Database, ChEBI, ChEMBL, MDDR) virtual screening. The results of this virtual screening are shown in Table 1, which contains a list of approved and experimental drugs, as well as other molecules that may be attractive for clinical and nonclinical applications.
[0144] Table 1:
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[0150] The ability of some of these ligands to induce a conformational switch of RBP4 was tested by expl...
Embodiment 3
[0155] Example 3: Integration of the RBP4-based LRPPI system into CAR
[0156] The third example demonstrates the applicability of LCN folding-based LRPPI in CAR function regulation by using the RBP4-based LRPPI system described in Example 1. Figure 6A , Figure 6B and Figure 9 The schematic of the CAR construct shown illustrates some of the testing possibilities for integrating this lipocalin fold-based LRPPI into a CAR. Figure 7A , Figure 7B , Figure 7C , Figure 7D and Figure 10 The sequences of the tested constructs are shown, Figure 8A , Figure 8B and Figure 8C shows the expression of signaling CAR constructs in primary human T cells. For each construct, primary human T cells were electroporated with 5 μg mRNA and tagged with Strep II 20 hours after electroporation, or in the "RS3CAR long, no c-myc tag" and "RF2 long CAR" CAR expression was detected by the Fc domain, using a biotinylated anti-human IgG1 antibody as the primary antibody and PE-conjugated s...
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