Part:BBa_K4623007

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:

Figure 1| Plasmid diagram of Cut Silinker 2

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.

Figure 2 |SDS-PAGE plot of IPTG concentration gradient induced expression of Cut Silinker 2 protein. The IPTG concentration gradient was set at 0, 0.1, and 0.25 mM at 37°C for 16h induced expression. Protein scale was compared with Blue Plus V Protein Marker at 10-190 kDa. The graphs showed lane bands in order of maker, 0 mM IPTG-induced bacterial sink, 0.1 mM IPTG-induced bacterial sink, 0.25 mM IPTG-induced bacterial sink, 0 mM IPTG-induced supernatant, 0.1 mM IPTG-induced supernatant, 0.25 mM IPTG-induced sediment, 0.1 mM IPTG-induced precipitation, and 0.1 mM IPTG-induced precipitation, and 0.25 mM IPTG-induced precipitation. It was electrophoresed at 80V for 30 min and 120V for 90 min successively and stained with Coomassie Brilliant Blue stain. There was no target band (18 kDa), but there was a clear band of about 36 kDa, and there was a possibility that this was due to the fact that CS2 was thermally unstable and dimerizes at 37°C.

Figure 3 |SDS-PAGE plot of IPTG concentration gradient induced expression of Cut Silinker 2 protein. The temperature conditions were changed to 16°C and IPTG concentration gradients were set to 0, 0.1, and 0.25 mM for 16 h of induced expression. Protein scale was compared with Blue Plus V Protein Marker at 10-190 kDa. The graphs showed lane bands in order of maker, 0 mM IPTG-induced bacterial sink, 0.1 mM IPTG-induced bacterial sink, 0.25 mM IPTG-induced bacterial sink, 0 mM IPTG-induced supernatant, 0.1 mM IPTG-induced supernatant, 0.25 mM IPTG-induced precipitation, 0.1 mM IPTG-induced precipitation, and 0.1 mM IPTG-induced precipitation, and 0.25 mM IPTG-induced precipitation.Successively electrophoresed at 80V for 30min and 120V for 90min, and stained with Coomassie Brilliant Blue stain. The appearance of target bands determined the expression temperature of 16°C.

Purification of Cut Silinker 2

Figure 4| CS2 purified bands (the last two lanes), it can be seen that the protein bands in the supernatant (flow-through solution) of CS2 induced expression after nickel column purification were not obviously missing, indicating that CS2 was not successfully bound to the nickel column, and the CS2 nickel column purification was invalid.

Structure and biological activity analysis

Structure simulation

In view of the difficulty of purification, we predicted the structure of the three parts of the Cut Silinker protein by alphafold2 and analyzed it.

Cut Silinker protein 2 presented an enveloped structural state, where the two parts of the intein overlap with each other wrapping around the possible enzyme cleavage site, and also possibly interfering with the ligand reaction, which was taken into account during the modeling process (see the modeling section for more details), but the His tag used for purification is relatively outwardly oriented, which should theoretically be advantageous for the purification, but it still had a some difficulty and may be influenced by other factors.

Figure 5 |Structure prediction results for Cut Silinker Protein 2, with blue color showing relatively high degree of fit and red color showing relatively low degree of fit, with medium confidence in between.

Enzyme digestion

Since protein purification was not successfully completed, this part of the results simulated the process using model, based on the concentration, mass, and relevant hydrophilic properties of the three proteins, as well as the Mie constants of the enzymatic reaction, and taking into account the temperature of the human body to simulate the release of the experimental drug as detailed in the [MODEL] section.

References

[1] Lun Yang, Jian Tang, Hui Yin, Jie Yang, Bin Xu, Yunkun Liu, Zhi Hu, Bentong Yu, Fangfang Xia, and Guowen Zou ACS Biomaterials Science & Engineering 2022 8 (2), 880-892 DOI: 10.1021/acsbiomaterials.1c01418.