Part:BBa_K4623008

Cut Silinker 3,SBP-linker

Usage and Biology

The C-terminal linker is a modified version of the Basic linker, divided into two parts while retaining the SBP portion, which interacts with mesoporous silica surfaces. Since the connection of arbitrary enzymatic cleavage sites requires the use of intein self-cleavage, the design of the CS linker includes two types of intein sequences. In the SBP-linker, the N-terminal of the NrdJ-C intein (BBa_K3308070) is connected to the N-terminus of the recombinant protein and can be linked to the C-terminus of the NrdJ-N intein (BBa_K3308069), present in the Cut linker. The design of NrdJ is based on the Pittsburg 2019 Intein Separation Logic Gate System (BBa_K3308007), which is found in our team’s parts library. NrdJ-C is essential for the self-splicing of the NrdJ intein and retains the functional splicing ability of the GP41-1 intein. It works in collaboration with GP41-1 to introduce the intermediate cleavage site into the silinker structure. The rigid linker and SBP are designed at the N-terminus of NrdJ-C, while the C-terminus of NrdJ-C includes a TEV recognition site and a HisTag.

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 CS3 is 8 kDa.

Plasmid diagram of Cut Silinker 3:

Figure 1| Plasmid diagram of Cut Silinker 3

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

We skipped this step by directly proceeding with the inducible expression purification of CS3 based on our experience with the inducible expression of CS1 and CS2.

Figure 2| CS3 4-20% Separation Gel SDS-PAGE.CS3 inducible expression system, see lane markers for induction conditions. It can be seen that there are obvious protein expression bands under each IPTG concentration induced expression conditions, but the molecular weight is different from that of CS3, and the induced expression faces difficulties.

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.

For Cut Silinker protein 3, the structure prediction was relatively low confidence, which was due to the fact that the SBP we chose belongs to the flexible structure itself, and its properties were subjected to a relatively large concentration of solution ions. In the actual experimental process, unfortunately we were not able to develop a solution favorable for its purification, which had a lot to do with our failure in purifying the process. However, we had taken into account the variability of this factor in the modeling process, so that the variability of its interaction in the linkage reaction and contacted with SiO2 had been simulated.

Figure 3 |Structure prediction results for Cut Silinker Protein 3, 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 our wiki [MODEL] section.