Subtilisin is a protease from Bacillus subtilis whose derivatives are being used in many washing detergents, which makes it suitable for a proof of principle.
As a serine protease it catalyses the break of a peptide bond by means of a catalytic triad consisting of serine 221, aspartic acid 32 and histidine 64. [1]
Figure 1: _____________ Previous studies prove, that replacing the serine with an alanine, which equals the loss of the reactive group, reduces the activity by factors of 2 x 10 ⁶. As the protection group would shield the hydroxyl group, it should be inhibiting in an at least this effective way. An orthogonal tRNA/synthetase pair that is able to introduce the photo-liable serine derivative O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine (DMNB-serine) at an amber stop, exists in Saccharomyces cerevisiae. It was developed and successfully used by P. G. Schulz [2]. DMNB-serine is converted to serine by irradiation with low energy blue light. The first sticking point is the production of subtilisin E in S. cerevisiae, for this an expression system has to be built. We chose to try three different approaches to compare efficiency of production, a plasmid with a constitutive promotor, one with an inducible and an inducible genome integration vector. Then by providing the yeast cells with the tRNA/synthetase pair for DMNB-serine and the respective unnatural amino acid can be incorporated.
Mutation of serine 221 to an amber stop codon in the subtilisin gene should lead to production of a photocaged and consequently inactive variant.
Also the naturally occurring secretion tag from B. subtilis, that allows relocation of the enzyme to the medium shall be exchanged with one working in S. cerevisiae, to make harvesting more convenient.
As previous efforts to express a protease from B. subtilis in S. cerevisiae, failed due to hyperglycosylation, [3] which resulted in an inactive enzyme, we also pursued a second strategy. Thus the idea to develop a DMNBS-synthetase that works in Escherichia coli evolved.

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.