Part:BBa_K4627043

Flexible site

To improve the performance of protein C we designed many protein C mutant genes. R8 is a complicated mutation of protein C, which contains10mutationsites:Q169L-H174Y-T192W-A193R-W194Y-T200L -V380Y- D625F-H627K-G629P

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2012
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 994
  Illegal AgeI site found at 1978
  Illegal AgeI site found at 2041
  
• 1000
  COMPATIBLE WITH RFC[1000]

Origin(organism)

Galleria mellonella

Structure Design

In our experiment, we searched for mutations that may form disulfide bonds through saturation mutations and predicted the structure of mutant C protein using AlphaFold v2.1.0. After identifying the mutant that forms disulfide bonds, we synthesized the primers we needed through GENEWIZ .

Figure 1. The mutant structure compared to the Wild type Ceres.

Molecular cloning

We first obtain wild-type Ceres from enterperise. pET22b(+) was the first vector we tried. Fortunately, it works. Then we used it as a template to clone different mutants.

Figure 2. The segment of C26 DNA is shown upwards.

• 1. Use mutated R8 primers to clone our small fragments..
  
• 2. Fuse the segments in a subsequent reaction by High-fidelity thermostable DNA polymerase.
  
• 3. Use Ligase to link our mutated short fragments and long fragments. .
  
• 4. Transform the constructed plasmid into competent DH5α cells to expand the plasmid largely
  
• 5. Extract the recombinant C26 sequence it to verify mutation sites
  

Expression in BL21 Escherichia coli

• 1. We expressed R8 in BL21 Escherichia coli and grew it in a 900ml large-scale system to obtain sufficient protein.
  
• 2. Use ultrasonic bacteria-breaking instrument to break bacteria and release the protein. After the crude protein extract was obtained through centrifugation, we used affinity chromatography to preliminarily purify the protein. SDS gel showed a distinct band between 75 and 65kDa with few stray bands. This indicated that relatively pure target protein was successfully obtained.

We used restriction endonuclease SalⅠ to linearize our recombinant plasmid.

• 3. After affinity chromatography, the protein was stored in the eluate buffer, which has a high imidazole concentration. Therefore, we replaced the solution with gel filtration chromatography buffer by desalting. It can be seen that cond peak is separated from protein peak.
  

Enzyme activity and thermostability determination

Tm is an important indicator of protein thermostability, called transition temperature. This value represents the temperature required for transition from native 3D conformation to unfolded state under certain conditions. We measured Tm of proteins by DSF. With the increase of temperature, the quaternary structure of protein was destroyed. Fluorescent dye binds to the exposed hydrophobic region and its fluorescence intensity changes. By measuring this change, Tm can be calculated. Tm of wild type Ceres was 46℃, and that of R8 reached 47℃, which was slightly increased.

• 1. Launch the Nanodrop software and select the "Nucleic Acid" function. After zeroing, add 1.5
  

µL of the sample and measure the protein concentration.

• 2. Prepare the reaction mix in PCR tubes according to Table 3. Set up three parallel sets for each
  

protein sample, adjusting the protein volume based on its concentration.

• 3. Gently mix the reaction mixture and place into the qPCR machine to initiate the reaction.
  
• 4.Process the data using Graphpad to obtain the Tm of the samples.