Part:BBa_K2020005

mutated expression system for subtilisin E in E. coli (Y77W)

Once introduced into Escherichia coli, this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. By performing a site-directed mutagenesis, tyrosine⁷⁷ in the propeptide of the enzyme was exchanged against tryptophan. Therefore, the enzyme loses its proteolytic activity.

For the varification of the function of this part, a skim milk assay on agar plates containing IPTG and the needed antibiotics was performed. Therefore, we streaked out the cells containing the modified expression systems on these plates and incubated at 30°C for three days.

The figure shows the skim milk plates assay. Cells containing the empty backbone (1) in comparison to either cells producing the native protease (2), cells with the SDM 1-modified (3) and the SDM 3-modified expression system (4) after incubation for 3 days at 30°C.

The empty backbone didn't cause a proteolytic activity. A clearance and therefore a proteolytic activity could be observed for the native protease (as demonstrated for the BioBrick [Part:BBa_K2020002|K2020002]) but also for the SDM 3 modified expression system. As SDM 3 had been executed to exchange tyrosine in the pro-peptide cleavage site against tryptophan, a proteolytic activity could be assumed caused by the clearance observed. Contrary to our former beliefs, it could now be deduced that exchanging tyrosine doesn’t result in a change of activity. Consequently, tyrosine in the pro-peptide cleavage site is not essential for the activity of subtilisin E. Unfortunately, we were not able to prove the principle of our project regarding tyrosine in the pro-peptide cleavage site, as it seems to be not essential for the proteolytic activity. Thus, exchanging tyrosine against a photo-labile, non-canonical amino acid more precisely ONB-tyrosine will not influence the activity of the enzyme.

Usage and Biology

Subtilisin E is an alkaline serine protease which non-specifically digests proteins. It is naturally produced by Bacillus subtilis.

This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for E. coli codon usage (BBa_K2020000).

Subtilisin E has to autoprocess itself to become functional. At first, the enzyme exists as a precursor, namely the pre-pro-subtilisin. The pre-sequence serves as a recognition sequence for secretion across the cytoplasmic membrane and is cleaved off in the course of the process. The pro-peptide acts as an intramolecular chaperone and facilitates the folding of the protease. Folding is essential for the activity of an enzyme. Still, the maturation process of Subtilisin E is not completed, as the pro-peptide covers the substrate binding site and inhibits activity. However, enough proteolytic activity is achieved to autoprocess the IMC-domain and therefore cleave off the pro-peptide. Yet, the C-terminal end of the pro-peptide continues to block the substrate binding site. After the degradation of the pro-peptide, the substrate-binding site is cleared and the protease becomes effectively active.

This mutated version of the BioBrick BBa_K2020002 was created to prove that tyrosine⁷⁷ in the propeptide cleavage-site of subtilisin E is essential for the activity of the enzyme.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 280
  
• 1000
  COMPATIBLE WITH RFC[1000]