Part:BBa_K4152101

AOX1 promoter+α-factor secretion signal+propeptide+PK_WT+His-Tag+AOX1 Terminator

This composite part is made up of 6 basic parts: AOX1 promoter, α-factor secretion signal, Propeptide, our target protein WT, the His-tag, and AOX1 terminator. These elements make our target protein better expressed and more convenient for subsequent protein detection.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2349
  Illegal XbaI site found at 1725
  Illegal PstI site found at 970
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2349
  Illegal PstI site found at 970
  Illegal NotI site found at 2361
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2349
  Illegal BamHI site found at 937
  Illegal XhoI site found at 1191
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2349
  Illegal XbaI site found at 1725
  Illegal PstI site found at 970
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2349
  Illegal XbaI site found at 1725
  Illegal PstI site found at 970
  Illegal AgeI site found at 2427
  
• 1000
  COMPATIBLE WITH RFC[1000]

Origin(organism)

Tritirachium album limber

Structure

Figure 1. Structure of wild-type Proteinase K (PDB ID=1ic6).

Molecular cloning

We used plasmids with Proteinase K (PK) synthesized by the company to do our next expression.

• 1. Use PK primers to clone our fragments which would be inserted into pPIC9.

Figure 2. Fragments of wild-type PK gene are PCR-amplified independently.

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

Figure 3. Double digestion of pPIC9 and wild-type PK.

• 3. Use Ligase to link our wild-type 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.

Figure 4. 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 5. 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 6. 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 wild-type 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 7. SDS-PAGE-1.

Figure 8. SDS-PAGE-2.

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.

Figure 9. Enzyme activity determination curve.

Conclusion

Wild-type Proteinase K shows poor thermostability at Room Temperature.