Part:BBa_K4152213

XhoⅠ+Linker a+propeptide+PK_MT13+His-Tag+Terminator+EcoRⅠ

These elements are the target PK gene we constructed, which would be used to insert the Proteinase K gene into pPIC9.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1159
  Illegal XbaI site found at 535
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1159
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1159
  Illegal XhoI site found at 4
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1159
  Illegal XbaI site found at 535
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1159
  Illegal XbaI site found at 535
  
• 1000
  COMPATIBLE WITH RFC[1000]

Origin(organism)

Tritirachium album limber

Structure Design

• 1. Use PyMOL to mutate some residues of Proteinase K, and analyze the possibility of the formation of new interaction forces like hydrogen bond, salt bond, disulfide bond, and π-π interaction.
• 2. Use AlphaFold v2.1.0 to predict the structure of the mutated PK.

Figure 1. The mutated PK structure compared to the Wild type PK.

• 3. Use FoldX to calculate the Gibbs Free Energy compared to wild-type PK with Ca²⁺. (PDB ID: 1ic6) The result of this mutated PK's ΔΔG is -13.19 kcal/mol.

Molecular cloning

We used the wild-type Proteinase K(Hereinafter referred to as PK) DNA gene to overlap our mutated PK gene.

Figure 2. The process of PCR for our mutated PK gene.

• 1. Clone this mutated PK by High-fidelity thermostable DNA polymerase based on part BBa_K4152211.

Figure 3. PCR Mutagenesis by Overlap Extension to obtain the mutated PK gene.

• 2. Use restriction endonuclease XhoⅠ and EcoRⅠ to double digest our mutated PK gene and pPIC9.

Figure 4. Double digestion of mutated PK.

• 3. Use Ligase to link our mutated PK and pPIC9 after double digestion.
  
• 4. Transform the constructed plasmid into competent DH5α cells to expand the plasmid largely
  
• 5. Extract the recombinant pPIC9-PK, verify it by double digestion (XhoⅠ and EcoRⅠ), and sequence it to verify mutation sites.

Figure 5. Double digestion verification of Recombinant pPIC9-PK.

After verification, it was determined that the construction is successful. We transformed the constructed plasmid into competent DH5α cells to expand the plasmid largely

Expression in Pichia Pastoris

Linearization of Recombinant pPIC9-PK:
We used restriction endonuclease SalⅠ to linearize our recombinant plasmid.

Figure 6. Linearization of Recombinant pPIC9-PK.

Electrotransformation:
Add several μg linearized pPIC9-PK to GS115 competence cells, then use a 1.5kV electric pulse to drill holes to let the gene get in.
Screen positive colonies and culture preservation:

• 1. Use MD solid medium to screen positive GS115 cells which can grow without Histidine. (Because GS115 cannot grow at medium without Histidine except our gene was introduced in).
  
• 2. Extract the genomic DNA of recombinant GS115 and verify the sequence of Recombinant pPIC9-PK (from AOX1 promoter to AOX1 Terminator, about 1500bp).

Figure 7. Genome PCR genomic DNA in Recombinant GS115.

• 3. Transfer the positive clones and preserve them in Glycerin (sterile), storing them at -80°C.
  

Express PK with Methanol:
Transfer some Glycerin recombinant GS115 to YPD, and culture overnight. Then transfer some YPD culture to BMG, culture overnight. Transfer some BMG culture to BMM, add 0.6% Methanol daily, express PK for several days, then collect the supernatant and concentrate it. At last, we do SDS-PAGE to make sure that the mutated PK has expressed successfully, and take the standard samples to do Western blot and quantitative analysis of stripes with ImageJ, then figure out the mass of PK.

Figure 8. SDS-PAGE-1.

Enzyme activity and thermostability determination

We use an Enzyme-labeled instrument to measure the Abs of OD_660nm of the product of L-Tyrosine of the reaction. We use 1% Casein as our substrate, and Tris-HCl (pH8.0) as our Buffer, and react at 55°C for several minutes. Then add trichloroacetic acid (TCA) to end the reaction, and centrifuge to collect the supernatant containing our L-Tyrosine product. Next step, we use Na₂CO₃ to provide the alkaline environment, then add the supernatant and Folin-phenol reagent to colorate L-Tyrosine. In the end, we detect the Abs of OD_660nm to assess the enzyme activity of our PK.
We store our PK at Room temperature for several days and detect the remains of it, then assess the thermostability of PK.

Conclusion

Maybe the mutated sites made our PK lose its enzyme activity.