Difference between revisions of "Part:BBa K4623007"

Revision as of 14:54, 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