Part:BBa_K4623006

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

Usage and Biology

Cut Silinker 1 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

figure1