Difference between revisions of "Part:BBa K3064024"
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<!-- Add more about the biology of this part here-->
 
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===Usage and Biology===
 
===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.  
+
Glucagon is a peptide hormone that generally elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis.[1]  
−
Trim21, an E3 ubiquitin-protein ligase functioning in the intracellular antibody-mediated proteolysis pathway,binds with high affinity to the Fc domain of antibody. HA Tag labels Trim21, making 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 after the translation.  
+
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 P2A mediated bicistronic expression of the recombinant glucagon-IgG-Fc and Trim 21 can be realized in cells. Glucagon binds to GCGR, Fc domain recruits the Trim21, leading to the ubiquitination and final degradation of GCGR.  
+
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 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.
+
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|]]
  
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<B/r>Figure 1.Representation of the function of the composite part.
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<B/r>Figure 1.Schematic epresentation of the function of the composite part.
 
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===Experimental Validation===
 
===Experimental Validation===
  
−
This part is validated for functional testing.
+
This part is validated for functional test.
  
−
For functional validation, we employed Western Blot, PAS dye, glucose Assay Kit and qPCR. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.
+
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://static.igem.org/mediawiki/parts/2/2f/T--NUDT_CHINA--2019_hepatocyte_dg.png
  
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[[File:MmuGlucagon-GGGGS-mmulgG-Fc-P2A-HA-mmuTrim21.jpg|400px|]]
+
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.
−
 
+
−
Figure 2. Functional evaluation of the GCGR degradation system in mouse primary hepatocytes after 24h of adenovirus infection.  
+
−
(A) Western Blot showing the result of glucagon receptor(GCGR) degradation. (B) PAS dye showing glycogenolysis variation. (C) Cell culture medium glucose concentration measured by glucose Assay Kit. (D) qPCR of crucial enzyme gene PEPCK and G6Pase in gluconeogenesis.
+
−
 
+
  
 +
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===
 
===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. BBa K3064024.png

Figure 1.Schematic epresentation of the function of the composite part.

Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 800
    Illegal BamHI site found at 1503
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 901
  • 1000
    INCOMPATIBLE 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.

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. (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.