Difference between revisions of "Part:BBa K4623007"

 
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<partinfo>BBa_K4623007 short</partinfo>
 
<partinfo>BBa_K4623007 short</partinfo>
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===Usage and Biology===
  
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Cuttable Silinker (CS) is a new recombinant protein, in addition to connecting to the surface of silica, the core breakthrough is the ability to cope with a variety of microenvironments specific cut, release off and then play a role in the release of the drugs. CS is divided into three parts, which can be self-sheared through the intein, and finally connected together to form a complete recombinant protein. The three components include: mSA-intein peptide (CS1) for coupling to the target protein, SBP-intein peptide (CS3) for linking to silica, and changeable recognized cleavage site (CS2). In our proof-of-concept, we chose the PLGVR motif, which can be recognized by metallo-matrix proteases (MMPs) in tumor microenvironments and added intein to both ends to achieve interchangeable insertion connections. that enable interchangeable insertion junctions.
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The Cut linker consists of the C-terminal sequence of the GP41-1 intein (BBa_K3308068), the N-terminal sequence of the NrdJ intein (BBa_K3308069), and a variable peptide segment. The C-terminal sequence of the GP41-1 intein is used to connect with the N-terminal linker (mSA-linker), while the N-terminal sequence of the NrdJ intein is used to connect with the C-terminal linker (SBP-linker). The variable peptide segment enables recognition and cleavage by protein cutting enzymes in specific environments, releasing the biotin-modified functional protein that has been cleaved and bound with mSA. To facilitate experimental verification, we have chosen a matrix metalloproteinase 2 (MMP2) cleavable short peptide (PLGVR) as the variable peptide segment. MMPs are a family of zinc-dependent endopeptidases that are overexpressed in the extracellular environment of certain tumors. The Cut Silinker, modified with PLGVR, can be recognized and cleaved by MMP2, releasing the functional protein. In addition, we have incorporated a TEV recognition site and a His tag for fusion protein purification[1].
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We determined the conditions for the production of His-tagged Cut Silinker by performing a small trial expression of the petDUT1 plasmid after transferring it into our engineered bacterium BL21(DE3). The purified Cut Silinker could be detected by SDS-PAGE, and the molecular weights of CS2 is 18 kDa.
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Plasmid diagram of Cut Silinker 2:
  
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===Usage and Biology===
 
  
 
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Revision as of 14:51, 11 October 2023


Cut Silinker 2, cutsite-linker, GP41-1-PLGVR-NrdJ

Usage and Biology

The Cut linker consists of the C-terminal sequence of the GP41-1 intein (BBa_K3308068), the N-terminal sequence of the NrdJ intein (BBa_K3308069), and a variable peptide segment. The C-terminal sequence of the GP41-1 intein is used to connect with the N-terminal linker (mSA-linker), while the N-terminal sequence of the NrdJ intein is used to connect with the C-terminal linker (SBP-linker). The variable peptide segment enables recognition and cleavage by protein cutting enzymes in specific environments, releasing the biotin-modified functional protein that has been cleaved and bound with mSA. To facilitate experimental verification, we have chosen a matrix metalloproteinase 2 (MMP2) cleavable short peptide (PLGVR) as the variable peptide segment. MMPs are a family of zinc-dependent endopeptidases that are overexpressed in the extracellular environment of certain tumors. The Cut Silinker, modified with PLGVR, can be recognized and cleaved by MMP2, releasing the functional protein. In addition, we have incorporated a TEV recognition site and a His tag for fusion protein purification[1].

We determined the conditions for the production of His-tagged Cut Silinker by performing a small trial expression of the petDUT1 plasmid after transferring it into our engineered bacterium BL21(DE3). The purified Cut Silinker could be detected by SDS-PAGE, and the molecular weights of CS2 is 18 kDa.

Plasmid diagram of Cut Silinker 2:


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]