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：

Figure 1| 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

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 Cut Silinker 1 protein expression induced by a gradient of 0.25 mM IPTG concentration at 16°C. Protein scale was compared with Blue Plus V Protein Marker at 10-190 kDa. The lane bands in the graph were maker, supernatant, bacterial sedimentation, and precipitation in that order. They were successively electrophoresed at 80V for 30 min and 120V for 90 min and stained with Coomassie Brilliant Blue stain. It appeared obvious target bands. Although most of them formed inclusion bodies, a certain amount of protein was still expressed in the supernatant.

Purification of Cut Silinker 1

After successfully obtaining the Cut silinker protein, it was necessary to culture and purify the target protein in large quantities. We induced expression on a large scale using 0.25mM IPTG and obtained a significant amount of the target protein after 16 hours of induction at 16℃. During the plasmid construction process, we had already attached a His tag to the target protein, which allows for purification using nickel column affinity chromatography based on the specificity of the His tag protein. Although inclusion bodies still formed, they could still meet the needs of subsequent experiments.

Figure 3|Cut Silinker 1 protein purification SDS-PAGE plot. Cut Silinker 1 mass expression was performed at 16°C and 0.25 mM IPTG concentration, followed by purification by nickel column affinity chromatography. The protein scale was Solarbio PR1930 Rainbow 245plus Broad Spectrum Protein Marker (5-245KD). Lanes 1-8 in the figure were rainbow marker, rainbow marker, CS1 supernatant, CS1 flow-through solution, 10 mM imidazole eluent, 40 mM imidazole eluent, 100 mM imidazole eluent, and 200 mM imidazole eluent, respectively. It was successively electrophoresed at 80v for 30min and 150v for 60min and stained with Coomassie Brilliant Blue staining solution. There were target bands in the supernatant and through-flow solution but not in the eluate.