Difference between revisions of "Team:Aachen/Description"
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Due to a limited range of tRNA/aminoacyl-synthetase pairs for non-canonical amino acids in general and especially for those that act orthogonally in <i>E. coli</i>, photocaging serine in the active site of subtilisin E with DMNB-serine is currently not possible. Hence, another strategy is needed to produce temporarily inactive proteases. This part of the project focuses on utilizing the maturation process of subtilisin E.<br/> | Due to a limited range of tRNA/aminoacyl-synthetase pairs for non-canonical amino acids in general and especially for those that act orthogonally in <i>E. coli</i>, photocaging serine in the active site of subtilisin E with DMNB-serine is currently not possible. Hence, another strategy is needed to produce temporarily inactive proteases. This part of the project focuses on utilizing the maturation process of subtilisin E.<br/> | ||
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| + | <table align="center" style=" border-collapse:separate; border: 0px ;border-spacing:20px;margin-left:20px;margin-right:80px; margin-top:0px;width: 910px; table-layout: fixed; "> | ||
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| + | <img src="xyz" style="position:relative;top:0px; left: 0px; "/> | ||
| + | <figcaption style="text-align: center; font-size:15px; "><b>Figure 1: maturation process of subtilisin E</b></figcaption> | ||
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| + | <p align="justify" style="padding-left: 1.0cm; padding-right: 1.0cm; font-size:16px;"> | ||
| + | Subtilisin E is an alkaline serine protease found in <i>Bacillus subtilis</i> that 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. [5] | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
Revision as of 10:25, 13 October 2016
Project Description
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Escherichia coli is widely used in synthetic biology. It offers the advantage of being a comparatively simple and well-understood model organism while being easy to handle in the laboratory environment. Also, an expansion of the genetic code has already been successfully implemented in E. coli multiple times [1]–[4] by introducing an orthogonal tRNA/aminoacyl-synthetase pair.
Therefore, working in E. coli is an obvious choice.
Due to a limited range of tRNA/aminoacyl-synthetase pairs for non-canonical amino acids in general and especially for those that act orthogonally in E. coli, photocaging serine in the active site of subtilisin E with DMNB-serine is currently not possible. Hence, another strategy is needed to produce temporarily inactive proteases. This part of the project focuses on utilizing the maturation process of subtilisin E.
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Subtilisin E is an alkaline serine protease found in Bacillus subtilis that 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. [5] |
