Part:BBa_K4152011

PK_MT11

To improve the performance of Proteinase K , we designed many Proteinase K mutant genes. PK_MT11 is a complicated mutation of Proteinase K, which contains 4 mutation sites: T16C-N257C-S17W-D260W.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 235
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 235
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 235
  
• 1000
  COMPATIBLE WITH RFC[1000]

Origin(organism)

Tritirachium album limber

Structure Design

• 1. We used PyMOL to mutate some residues of Proteinase K, analyse the possibility of the formation of new interaction force like hydrogen bond, salt bond, disulfide bond, π-π interaction.
• 2. Then we used AlphaFold to predict the structure of our mutated PK.

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

• 3. Finally, we used FoldX to calculate the Gibbs Free Energy compared to wild type PK. (PDB ID: 1ic6) The result of this mutated PK's ΔΔG is -13.81kcal/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. We used mutated PK primers to clone our small fragments.

Figure 3. The first time PCR for our small fragments-1.

Figure 4. The first time PCR for our small fragments-2.

• 2. We overlapped the small fragments by High-fidelity thermostable DNA polymerase.

Figure 5. The overlap PCR for our entire PK fragment.

• 3. Use restriction enzyme XhoⅠ and EcoRⅠ to double digest our mutated PK gene and pPIC9.

Figure 6. Double digestion of mutated PK.

Figure 7. Double digestion of pPIC9.

• 4. Use Ligase to link our mutated PK and pPIC9 after double digestion.
  
• 5. Then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
  
• 6. Extract the recombinant pPIC9-PK and verify it by double digestion (XhoⅠ and EcoRⅠ), and sequence it for verication of mutation sites.

Figure 8. Double digestion verification of Recombinant pPIC9-PK.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli DH5α to expand the plasmid largely.

Expression in Pichia Pastoris

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

Figure 9. Linearization of Recombinant pPIC9-PK.

Electrotransformation:
Add several μg linearized pPIC9-PK to GS115 competence cells, then use 1.5kV electric pulse to drill holes to let 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 genome of recombinant GS115 and verify the sequence of Recombinant pPIC9-PK (from AOX1 promoter to AOX1 Terminator, about 1500bp).

Figure 10. Genome PCR verification of Recombinant GS115.

• 3. transfer the positive clonies and preserve it in Glycerin (steriled), store it at -80°C.
  

Express PK with Methanol:
Transfer some Glycerin recombinant GS115 to YPD, 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 concentration it. At last, we do SDS-PAGE to make sure that our PK has expressed successfully, and take the standard samples to do grey analysis, then figure out the mass of PK.

Figure 11. SDS-PAGE-1.

Figure 12. SDS-PAGE-2.

Enzyme activity and thermostability determination

We use ELIASA 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, react at 55°C for several minutes. Then add trichloroacetic acid (TCA) to end the reaction, centrifuge to collect the supernatant where contains our product of L-Tyrosine. Next step, we use Na₂CO₃ to provide alkaline environment, then add the supernatant and Folin-phenol reagant to colorate L-Trosine. 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.

Figure 13. Enzyme activity determination, compared with wild type.

Conclusion

In conclusion,the thermostability of PK_MT11 has improved 在这里填东西 times, compared with WT(wild type).