Team:TU Eindhoven/Background/Orthogonal

Orthogonal tRNA System

Normally, all proteins produced by a cell are built with the 20 amino acids naturally present in cells. However, protein engineering has made the incorporation of unnatural amino acids possible. This is done by means of an orthogonal tRNA aminoacyl-tRNA synthetase and tRNA pair. An orthogonal system does not interfere with any other system in the cell. The anticodon on the tRNA corresponds with the TAG codon (amber stop codon). This codon has to be placed in the gene of interest to incorporate the unnatural amino acid.

The aminoacyl-tRNA synthetase is specifically modified to bind the unnatural amino acid to the orthogonal tRNA. The tRNA, in response to an amber stop codon(UAG) on the mRNA, incorporates the unnatural amino acid into the amino acid sequence. (Figure 1) [1]

Figure 1. The incorporation of an unnatural amino acid into a protein.

Unnatural Amino Acid p-Azido-L-Phenylalanine

The unnatural amino acid used to incorporate an azide in the anchor proteins is p-Azido-L-phenylalanine (pAzF). pAzF is a photocrosslinker which can be incorporated in any protein, irrespective of its size or sequence, by a tRNA synthetase/tRNA pair and the amber codon TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslinks interacting proteins.

Both CPX and INPNC meet the requirements for an anchor protein. They are able to display certain unnatural amino acids (in this case pAzF) on the outside of the cell, where the reaction with the DBCO-conjugate takes place.[2]

Bibliography

[1]Davis, Lloyd, and Jason W. Chin.(2012) "Designer proteins: applications of genetic code expansion in cell biology."Nature Reviews Molecular Cell Biology 13 : 168-182.

[2]Chin, Jason W., Stephen W. Santoro, Andrew B. Martin, David S. King, Lei Wang, and Peter G. Schultz. (2002) "Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli." J. Am. Chem. Soc. 124.31 : 9026-9027