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/synthetase pair.
Therefore, working in E. coli is an obvious choice.

Due to a limited range of tRNA/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.

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]

This mechanism can be used to implement a novel inactivation method.

O-(2-Nitrobenzyl)-L-tyrosine (ONBY) is a derivate of the canonical amino acid tyrosine. It carries a photo-labile protection group that can be cleaved off by irradiation with UV-light (365nm, [6]).
	By adding ONBY to the genetic code of E. coli and incorporating said amino acid in the pro-peptide cleavage-site of subtilisin E the maturation process is disturbed. Due to its size ONBY sterically hinders the protease [7]. The pro-peptide cannot be cleaved from the enzyme and subtilisin E is not able to achieve its full proteolytic activity. A temporarily inactive protease is produced.

After removal of the protection group the maturation process can be completed and subtilisin E acquires its full proteolytic activity.

References

[1]  J. W. Chin, A. B. Martin, D. S. King, L. Wang, and P. G. Schultz, “Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli,” Proc. Natl. Acad. Sci., vol. 99, no. 17, pp. 11020–11024, 2002.
[2]  J. W. Chin, “Reprogramming the genetic code,” EMBO J., vol. 30, no. 12, pp. 2312–2324, 2011.
[3]  R. A. Mehl et al., “Generation of a Bacterium with a 21 Amino Acid Genetic Code,” J. Am. Chem. Soc., vol. 125, no. 4, pp. 935–939, Jan. 2003.
[4]  L. Wang and P. G. Schultz, “Expanding the Genetic Code,” Angew. Chem. Int. Ed., vol. 44, no. 1, pp. 34–66, Jan. 2005.
[5]  X. Fu, M. Inouye, and U. Shinde, “Folding Pathway Mediated by an Intramolecular Chaperone,” J. Biol. Chem., vol. 275, no. 22, pp. 16871–16878, 2000.
[6]  M. S. Kim and S. L. Diamond, “Photocleavage of o-nitrobenzyl ether derivatives for rapid biomedical release applications,” Bioorg. Med. Chem. Lett., vol. 16, no. 15, pp. 4007–4010, Aug. 2006.
[7]  C. Chou, D. D. Young, and A. Deiters, “A Light-Activated DNA Polymerase,” Angew. Chem. Int. Ed., vol. 48, no. 32, pp. 5950–5953, Jul. 2009.