Difference between revisions of "Part:BBa K4623007"

Revision as of 14:56, 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

Cultivation, Purification and SDS-PAGE

Induction condition

The presence of mSA monomers will easily cause the protein to form inclusion bodies and increase the difficulty of purification. In order to make our Cut Silinker express efficiently and reduce the generation of inclusion bodies, we screened the IPTG-induced expression conditions. We set five IPTG concentration gradients, namely: 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, and the final results of protein expression showed that the concentration of 0.25 mM was the best. In addition, we set two temperature gradients of 37℃ induced expression and 16℃ induced expression. 37℃ protein formed inclusion bodies instead of soluble protein in large quantities, so we finally determined 16℃ as the effective induction temperature.

In order to make mSA fold normally and reduce the generation of inclusion bodies, we modified the buffer of protein by adding biotin. The combination of biotin and mSA could help the Cut silinker protein fold normally and reduce the inclusion bodies formed after misfolding. Finally, we got the soluble protein that could be extracted in the supernatant. The formula of the buffer and the experimental procedure could be found in our wiki.