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

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(50μg/mL) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 900ml LB medium and added antibiotic to 50 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 5-6 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-His tag fused with target protein.

• First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  
• Second, add the protein solution to the column, let it flow naturally and bind to the column.
  
• Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  
• Forth,add Ni-Washing buffer several times. Check as above. Collect the eluted proteins for further operation.
  

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).