Part:BBa_K4623006

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

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.

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.

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

figure1