Difference between revisions of "Part:BBa K3064024"
(→Experimental Validation) |
(→Experimental Validation) |
||
| Line 30: | Line 30: | ||
This part is validated for functional test. | This part is validated for functional test. | ||
| − | This part was transported into mouse primary hepatocytes by adenovirus. | + | This part was transported into mouse primary hepatocytes by adenovirus.to validate the function of this part, co-IP and Western Blot were performed. Results were shown below. |
https://2019.igem.org/wiki/images/9/96/T--NUDT_CHINA--western.jpg | https://2019.igem.org/wiki/images/9/96/T--NUDT_CHINA--western.jpg | ||
| − | Figure 2. Fuctional validation of the GCGR degradation system(parts BBa_K3064024) in mouse primary hepatocytes 24h | + | Figure 2. Fuctional validation of the GCGR degradation system(parts BBa_K3064024) in mouse primary hepatocytes 24h after adenovirus infection. Western Blot showing the degradation of GCGR. |
This part expresses GCGR binds with glucagon and these three parts form a ternary complex. When trim21 functions as an E3 ubiquitin ligase, the complex would be ubiquitin tagged and degraded through the proteasome pathway. In this way, intracellular GCGR content in hepatocytes would decrease, and so would those distributed in cell membranes. | This part expresses GCGR binds with glucagon and these three parts form a ternary complex. When trim21 functions as an E3 ubiquitin ligase, the complex would be ubiquitin tagged and degraded through the proteasome pathway. In this way, intracellular GCGR content in hepatocytes would decrease, and so would those distributed in cell membranes. | ||
Revision as of 19:24, 21 October 2019
mmuGlucagon-GGGGS-mmulgG-Fc-P2A-HA-mmuTrim21
This part is an effective tool for the degradation of glucagon receptor (GCGR).
Usage and Biology
Glucagon is a peptide hormone that generally elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis.[1] Immunoglobulin G (IgG) is a type of antibody, of which fragment crystallizable region (Fc) is the tail region. Trim21, an E3 ubiquitin-protein ligase functioning in the intracellular antibody-mediated proteolysis pathway,binds with high affinity to the Fc domain of antibody. Trim21 was labeled with HA-tag, which makes it easier to be detected by Western Blotting. P2A is a member of 2A peptides family that can be used to cleave a longer peptide into two shorter peptides during the translation process.
With this composite part,the bicistronic expression of the recombinant glucagon-IgG-Fc and HA-Trim21 could be achieved in mammalian cell. with Glucagon binding to GCGR and Fc domain recruiting the Trim21, endogenous GCGR would be labeled with ubiquitins and subsequently degradated via endogenous ubiquitin-proteasome pathway.
Special Design
Special designs were taken to optimize the applicability and adaptive of such parts.We disassembled the sequence of IgG and fused it with glucagon to get the recombinant glucagon-IgG-Fc.HA Tag was added to N-terminal of Trim21.The two functional modules of the part (glucagon-IgG-Fc and Trim 21) were connected by the linker GGGGS,one of the best fold breaking linkers to expose fusion protein partner well.
Figure 1.Representation of the function of the composite part.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 800
Illegal BamHI site found at 1503 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 901
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 125
Experimental Validation
This part is validated for functional test.
This part was transported into mouse primary hepatocytes by adenovirus.to validate the function of this part, co-IP and Western Blot were performed. Results were shown below.
Figure 2. Fuctional validation of the GCGR degradation system(parts BBa_K3064024) in mouse primary hepatocytes 24h after adenovirus infection. Western Blot showing the degradation of GCGR.
This part expresses GCGR binds with glucagon and these three parts form a ternary complex. When trim21 functions as an E3 ubiquitin ligase, the complex would be ubiquitin tagged and degraded through the proteasome pathway. In this way, intracellular GCGR content in hepatocytes would decrease, and so would those distributed in cell membranes.
The highlight of this part is that the glucagon in this part can be replaced. It could be an antibody, or it could be another ligand, or else. Almost any protein is possible to be directly degraded in cell if the part expresses some fragments that could binds with the target.
References
1. Voet D, Voet JG (2011). Biochemistry (4th ed.). New York: Wiley.