Difference between revisions of "Part:BBa K3064024"
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===Special Design=== | ===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 | + | Special designs were taken to optimize the applicability and adaptive of such parts. We disassembled the Fc sequence of IgG and fused it with glucagon to obtain the recombinant Glucagon-IgG-Fc. HA-tag was added to N-terminal of Trim21 to make it easier to be detected by Western Blotting.The two functional modules of the part (Glucagon-IgG-Fc and Trim21) were connected by the linker GGGGS,one of the most flexible peptide linkers to guarantee that two parts function independently. |
[[File:BBa_K3064024.png|500px|]] | [[File:BBa_K3064024.png|500px|]] | ||
| − | <B/r>Figure 1. | + | <B/r>Figure 1.Schematic epresentation of the function of the composite part. |
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This part is validated for functional test. | 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. | + | This part was transported into mouse primary hepatocytes by adenovirus for a closer mimic to the in vivo application condition.to validate the function of this part, co-IP and Western Blot were performed. Results were shown below. |
| − | https:// | + | https://static.igem.org/mediawiki/parts/2/2f/T--NUDT_CHINA--2019_hepatocyte_dg.png |
| − | 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. | + | Figure 2. Fuctional validation of the GCGR degradation system(parts BBa_K3064024) in mouse primary hepatocytes 24h after adenovirus infection. (A) Western Blot showing the significant degradation of GCGR. (B) Greyscale analysis was performed to quantify the expression of GCGR in mouse primary hepatocytes.Relative protein expression levels were calculated by using β-actin expression level as normalization and then set the protein expression level in the control groups arbitrarily as 1.0. Error bar represents SD of at least 3 biological replicates, ** p<0.01. |
| − | + | The Glucagon-IgG Fc and HA-Trim21 expressed by this parts would bind with GCGR and Fc respectively. These three parts would form a ternary complex, while Trim21 functions as an E3 ubiquitin ligase. Then, the complex would be ubiquitinated and degraded via the ubiquitin-proteasome pathway. Therefore, intracellular GCGR would decrease, and so would those distributed in cell membranes during the turnover of membrane protein. | |
| − | The highlight of this part is that the glucagon in this part can be replaced. It could be | + | The highlight of this part is that the glucagon in this part can be replaced. It could be a single domain antibody, a single chain antibody, a peptide aptamer, a ligand, or any peptide module capable of specifically interacting with selected targets. Therefore, almost every protein is possible to be directly degraded in mammanlian cell if glucagon in this part were replaced with some fragments that could bind with the target specifically, which means this part has very good scalability and application prospect. |
===References=== | ===References=== | ||
Latest revision as of 03:58, 22 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 Fc sequence of IgG and fused it with glucagon to obtain the recombinant Glucagon-IgG-Fc. HA-tag was added to N-terminal of Trim21 to make it easier to be detected by Western Blotting.The two functional modules of the part (Glucagon-IgG-Fc and Trim21) were connected by the linker GGGGS,one of the most flexible peptide linkers to guarantee that two parts function independently.
Figure 1.Schematic epresentation 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 for a closer mimic to the in vivo application condition.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. (A) Western Blot showing the significant degradation of GCGR. (B) Greyscale analysis was performed to quantify the expression of GCGR in mouse primary hepatocytes.Relative protein expression levels were calculated by using β-actin expression level as normalization and then set the protein expression level in the control groups arbitrarily as 1.0. Error bar represents SD of at least 3 biological replicates, ** p<0.01.
The Glucagon-IgG Fc and HA-Trim21 expressed by this parts would bind with GCGR and Fc respectively. These three parts would form a ternary complex, while Trim21 functions as an E3 ubiquitin ligase. Then, the complex would be ubiquitinated and degraded via the ubiquitin-proteasome pathway. Therefore, intracellular GCGR would decrease, and so would those distributed in cell membranes during the turnover of membrane protein.
The highlight of this part is that the glucagon in this part can be replaced. It could be a single domain antibody, a single chain antibody, a peptide aptamer, a ligand, or any peptide module capable of specifically interacting with selected targets. Therefore, almost every protein is possible to be directly degraded in mammanlian cell if glucagon in this part were replaced with some fragments that could bind with the target specifically, which means this part has very good scalability and application prospect.
References
1. Voet D, Voet JG (2011). Biochemistry (4th ed.). New York: Wiley.