Difference between revisions of "Part:BBa K5301000"
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Nanodisc technology is a widely applicable approach to render membrane proteins soluble in aqueous solutions in a native-like bilayer environment, where the membrane proteins remain stable and active. The Nanodisc is a non-covalent structure of phospholipid bilayer and membrane scaffold protein (MSP), a genetically engineered protein, which mimics the function of Apolipoprotein A-1 (ApoA-1). The first MSP, MSP1, was engineered with its sequence based on the sequence of A-1, but without the globular N-terminal domain of native A-1. The MSP1E3D1 variant of MSP1 differs from MSP1 in the following facets: (1) It deletes the first 11 amino acids in the Helix 1 portion of the original MSP1 sequence (which is known separately as MSP1D1). The MSP1D1 protein is an N-terminal histidine-tagged protein with a TEV protease cleavage site between the histidine-tag and the protein sequence. (2) It repeats the Helix 4 (H4), Helix 5 (H5) and Helix 6 (H6) sequences of the original MSP1 sequence between the parent Helix 6 (H6) and Helix 7 (H7) segments of MSP1D1.
 
Nanodisc technology is a widely applicable approach to render membrane proteins soluble in aqueous solutions in a native-like bilayer environment, where the membrane proteins remain stable and active. The Nanodisc is a non-covalent structure of phospholipid bilayer and membrane scaffold protein (MSP), a genetically engineered protein, which mimics the function of Apolipoprotein A-1 (ApoA-1). The first MSP, MSP1, was engineered with its sequence based on the sequence of A-1, but without the globular N-terminal domain of native A-1. The MSP1E3D1 variant of MSP1 differs from MSP1 in the following facets: (1) It deletes the first 11 amino acids in the Helix 1 portion of the original MSP1 sequence (which is known separately as MSP1D1). The MSP1D1 protein is an N-terminal histidine-tagged protein with a TEV protease cleavage site between the histidine-tag and the protein sequence. (2) It repeats the Helix 4 (H4), Helix 5 (H5) and Helix 6 (H6) sequences of the original MSP1 sequence between the parent Helix 6 (H6) and Helix 7 (H7) segments of MSP1D1.
  
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===Experimental Design and Results===
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Our ultimate goal is to successfully construct a well-functioning nanoplate, thus it is crucial to explore the construction process and identify suitable conditions for the nanoplate. Literature has already demonstrated that MSP1E3D1 can successfully construct nanoplates [1], therefore, we have decided to use MSP1E3D1 as our target protein to explore the nanoplate construction process suitable for our experimental conditions.
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We found the pMSP1E3D1 plasmid on Addgene, which can express MSP1E3D1 in E. coli. After transferring the pMSP1E3D1 plasmid into E. coli BL21 (DE3) cells, we obtained single colonies. PCR was performed on the single colonies, and the results were verified by agarose gel electrophoresis (Figure 1). Among the four selected single colonies, colony 4 most successfully obtained the target band.
 
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===Usage and Biology===
 
===Usage and Biology===

Revision as of 11:18, 30 September 2024


MSP1E3D1 is a genetically engineered protein, which mimics the function of ApoA-I.

Usage and Biology

Nanodisc technology is a widely applicable approach to render membrane proteins soluble in aqueous solutions in a native-like bilayer environment, where the membrane proteins remain stable and active. The Nanodisc is a non-covalent structure of phospholipid bilayer and membrane scaffold protein (MSP), a genetically engineered protein, which mimics the function of Apolipoprotein A-1 (ApoA-1). The first MSP, MSP1, was engineered with its sequence based on the sequence of A-1, but without the globular N-terminal domain of native A-1. The MSP1E3D1 variant of MSP1 differs from MSP1 in the following facets: (1) It deletes the first 11 amino acids in the Helix 1 portion of the original MSP1 sequence (which is known separately as MSP1D1). The MSP1D1 protein is an N-terminal histidine-tagged protein with a TEV protease cleavage site between the histidine-tag and the protein sequence. (2) It repeats the Helix 4 (H4), Helix 5 (H5) and Helix 6 (H6) sequences of the original MSP1 sequence between the parent Helix 6 (H6) and Helix 7 (H7) segments of MSP1D1.

Experimental Design and Results

Our ultimate goal is to successfully construct a well-functioning nanoplate, thus it is crucial to explore the construction process and identify suitable conditions for the nanoplate. Literature has already demonstrated that MSP1E3D1 can successfully construct nanoplates [1], therefore, we have decided to use MSP1E3D1 as our target protein to explore the nanoplate construction process suitable for our experimental conditions.

We found the pMSP1E3D1 plasmid on Addgene, which can express MSP1E3D1 in E. coli. After transferring the pMSP1E3D1 plasmid into E. coli BL21 (DE3) cells, we obtained single colonies. PCR was performed on the single colonies, and the results were verified by agarose gel electrophoresis (Figure 1). Among the four selected single colonies, colony 4 most successfully obtained the target band. Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 115
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 115
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 115
    Illegal BglII site found at 741
    Illegal XhoI site found at 637
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 115
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 115
  • 1000
    COMPATIBLE WITH RFC[1000]