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

Molecular cloning

We used the wild type Proteinase K DNA gene to overlap our mutated PK gene.

• 1. We used mutated PK primers to clone our small fragments.

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

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

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

Figure 3. 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 4. Double digestion of mutated PK.

Figure 5. 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 6. 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 7. 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:

• First, 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.
  
• Second, extract the genome of recombinant GS115 and verify the sequence of Recombinant pPIC9-PK (from AOX1 promoter to AOX1 Terminator, about 1500bp).

Figure 7. Genome PCR verification of Recombinant GS115.

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

Figure 2. The result of SDS-PAGE.

Gel filtration chromatography:
The collected protein samples are concentrated in a 30 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 75 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

Figure 3. The result of gel filtration used the superdex75 column with the AKTA system.

Enzyme activity determination

We use HPLC equipment to measure the peak area of the product of PET(MHET) of the reaction, in order to express the enzyme activity of PETase. For more information on the product of PET(MHET), please see our project introduction.

Figure 4. Enzyme activity determination, compared with wild type.

Conclusion

In conclusion,the enzyme activity and thermostability of Super5 has greatly improved 163 times ，compared with WT(wild type).