Part:BBa_K2027000

Recombinant Apoptosis-Inducing Protein (L-lysine Alpha Oxidase)

This part is a native Scomber japonicus enzyme codon-optimized for Escherichia coli. The conjugated tag should allow for purification and visualization with anti-FLAG antibodies, visualization with Lumio™ Green, and purification with nickel columns. Tani et al. characterized this enzyme in their quest for synthesis of L-pipecolic acid from racemic lysine after nickel column purification, and we verified the function of our part in a similar way. More information on the function of the enzyme is located in the Experience page.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1004
  Illegal BglII site found at 1032
  Illegal BamHI site found at 767
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 25
  

Characterization

We were able to transform this part into E. coli and induce protein synthesis using IPTG. We extracted this protein by relying on its HIS-tag and using Ni-NTA resin columns.

We ran a SDS-PAGE gel and used Lumio-Green staining on the fractions we collected during the protein purification process to verify which fraction contained the isolated protein. In the gel below, the lanes, from left to right, become progressively cleaner and display a singular prominent band. The smear/spread may be due to extraneous cell content that remained in the cell lysate and that had not yet been removed.

After determining the protein existed in the elution buffer, we continued with a large-scale protein extraction and purification. With our final product, we wanted to measure its purity. Using the bicinchoninic acid assay (BCA) to determine the total concentration of protein in our final elution buffer, we found that our purified protein had a concentration of 149.317 mg/mL (shown below from Nanodrop 2000 software).