Difference between revisions of "Part:BBa K4623006"
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<partinfo>BBa_K4623006 short</partinfo>
 
<partinfo>BBa_K4623006 short</partinfo>
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===Usage and Biology===
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1. **Usage and Biology**
  
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Cut 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, BBa_K4623007). 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 N-terminal linker is a modified version of the Basic linker, divided into two parts while retaining the mSA portion, and still plays a role in constructing an avidin-biotin affinity system. The C-terminus of the recombinant protein contains a segment with the N-terminal sequence of GP41-1 (BBa_K3308067), which can be connected to the N-terminal of GP41-1C of the Cut linker (BBa_K3308068). Additionally, a TrxA solubility-enhancing protein tag is added to the N-terminus to improve protein solubility for expression. The His-tag is used for purification and separation, while the thrombin site is employed to remove the tag after purification.
  
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The CS1 is a modified version of the Basic Silinker, divided into two parts while retaining the mSA portion, and still plays a role in constructing an avidin-biotin affinity system. The C-terminus of the recombinant protein contains a segment with the N-terminal sequence of GP41-1 (BBa_K3308067), which can be connected to the N-terminal of GP41-1C of the Cut linker (BBa_K3308068). Additionally, a TrxA solubility-enhancing protein tag is added to the N-terminus to improve protein solubility for expression. The His-tag is used for purification and separation, while the thrombin site is employed to remove the tag after purification.
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After CS1 protein expression, cleavage of thrombin（part number) allowed for exposure of the mSA (part number) site to bind to functional proteins that had been biotinylated.  
  
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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 CS1 is 41 kDa.
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===Usage and Biology===
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Plasmid diagram of Cut Silinker 1：
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 14:10, 11 October 2023


Cut Silinker 1(mSA-linker), TrxA-His-thrombin-mSA-GP41-1

Usage and Biology

1. **Usage and Biology**

The N-terminal linker is a modified version of the Basic linker, divided into two parts while retaining the mSA portion, and still plays a role in constructing an avidin-biotin affinity system. The C-terminus of the recombinant protein contains a segment with the N-terminal sequence of GP41-1 (BBa_K3308067), which can be connected to the N-terminal of GP41-1C of the Cut linker (BBa_K3308068). Additionally, a TrxA solubility-enhancing protein tag is added to the N-terminus to improve protein solubility for expression. The His-tag is used for purification and separation, while the thrombin site is employed to remove the tag after purification.

After CS1 protein expression, cleavage of thrombin（part number) allowed for exposure of the mSA (part number) site to bind to functional proteins that had been biotinylated.

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 CS1 is 41 kDa.

Plasmid diagram of Cut Silinker 1： 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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 445
    Illegal AgeI site found at 505
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 909

Cultivation, Purification and SDS-PAGE

Induction Condition

图片描述

figure1