Part:BBa_K4016003
LD3
This part interacts with Bcl-xl(Part:BBa_K4016002) through the induction of small molecule. Technically, we use A1331852 and A1155463 to induce the dimerization of this part and Bcl-xl. Functionally, we use this pair to fuse with other proteins respectively, bringing our system together in a controllable way.
Usage and Biology
LD3 is a human apolipoprotein E4 which shows to have a good bond with Bcl-XL. In the past report, 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.
Fusing BcL-XL and LD3 fragment to the Trim21 and its targeting module, we can realize the Trim21-induced degradation on it’s target protein, and the BcL-XL-LD3 dissociation under A1331852 can induced the dissociation of Trim21 and its targeting module, so as to stop the degradation.
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.
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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