Part:BBa_K2020006
mutated expression system for subtilisin E in E. coli (Y77X)
Once introduced into Escherichia coli, this BioBrick is able to produce a modified 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 the amber stop codon UAG. Therefore, the expression of the enzyme is interrupted.
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 integrate a non-canonical amino acid into the sequence of the propeptide of subtilisin E by adding an orthogonal tRNA/aminoacyl-synthetase pair that is capable of incorporating the non-canonical amino acid of choice in response to the UAG stop codon.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 280
- 1000COMPATIBLE WITH RFC[1000]
//chassis/prokaryote/ecoli
biology | Bacillus subtilis |
chassis | Escherichia coli |
protein | subtilisin E |