Part:BBa_K2653000
trim21
Trim21 is a type of E3 ubiquitin ligase that has been discovered in many eukaryotic cells.
Usage and Biology
The Tripartite motif-containing protein 21(Trim21) belongs to the E3 ubiquitin ligase family, which is encoded by the TRIM21 gene.The C-terminal B30.2 domain on trim21 shapes like a crab and offers a site for the conservative Fc region of human IgG 1,2 and 4 to bind with. This makes trim21 a potential protein degradation tool---when it binds with hIgG1-Fc, and after the antibody-antigen interaction, a ternary trim21-antibody-antigen complex would be built up. The trim21 then functions as a E3 ubiquitin ligase and proceeds the complex to be depleted through the ubiquitin-proteasome pathway.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 204
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 873
Illegal BamHI site found at 1411 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 161
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1251
Source
The sequence of trim21 comes from the article: James L C, Keeble A H, Khan Z, et al. Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(15):6200-6205.
Experimental Validation
Sequencing
This part is sequenced as correct after construction.
PCR
Methods
The PCR is performed with Premix EX Taq by Takara.
F-Prime: 5’- aatccttagctttcgctaaggatgatttctg-3’
R-Prime: 5’- ccttgcccttttttgccgga-3’
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.
Figure 2. Electrophoresis showing the production of four miRNA lockers by asymmetric PCR.
Enzyme digestion test
Methods
After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with OMEGA e.Z.N.A Plasmid Mini Kit I(200). The cutting procedure was performed with EcoRI and SpeI restriction endonuclease bought from TAKARA.
The plasmid was cutted in a 10μL system at 37 ℃ for 4 hours. The Electrophoresis was performed on a 1％ Agarose glu.
The result of the agarose electrophoresis was shown on the picture above.
Functional Test
Source
The sequence of trim21 comes from the article: James L C, Keeble A H, Khan Z, et al. Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(15):6200-6205.
References
1]Clift D, Mcewan W A, Labzin L I, et al. A Method for the Acute and Rapid Degradation of Endogenous Proteins[J]. Cell, 2017, 171(7):1692-1706.e18. [2]Mcewan W A, Falcon B, Vaysburd M, et al. Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(3):574.
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