Part:BBa_K4016002
Bcl-xl
This part interacts with LD3(Part:BBa_K4016003) through the induction of small molecule. Technically, we use A1331852 and A1155463 to induce the dimerization of this part and LD3. Functionally, we use this pair to fuse with other proteins respectively, bringing our system together in a controllable way.
Usage and Biology
B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic Bcl-2 protein found in the mitochondrial membrane. MotifGraft performs extensive structural searches in protein databases to identify scaffolds with backbone simi-larity to a binding motif, as well as structural compatibility to a given binding partner. Finally human apolipoprotein E4 (LD3) shows to have a good bond with Bcl-XL.
Two known small molecules, A1331852 and A1155463, have been reported to bind to Bcl-XL at less than 10 pM and were shown by SPR and size-exclusion chromatogra-phy coupled to a multi-angle light scatter (SEC-MALS) to dissoci-ate Bcl-XL from LD3, with an apparent half-maximum inhibitory concentration (IC50) of 242 nM and 101 nM , respectively.
Characterization
This part BBa_K4016024 was cloned in pXQ109 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.This part is validated through four ways: PCR, Sequence, and functional testing.
PCR
The PCR is performed with Premix EX Taq.
F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGgagtctgggggag 3’（oXQ218 forward prime）
R-Prime：5’gctgtagtccaggatTCCGTACAGTTCCACGAAGGT3’（oXQ169 reverse prime）
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.
Sequence
This part is sequenced as correct after construction.
Experimental Validation
Result
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
[1] Giordano-Attianese, G. et al. A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nat Biotechnol 38, 426–432 (2020).
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