Non-canonical amino acids

A.J. Link, M.L. Mock, D.A. Tirrell, Non-canonical amino acids in protein engineering, Curr. Opin. Biotechnol. 14(6) (2003) 603-609. 

Although such nanotubes form an interesting structure with potential for exterior functionalization, they are currently limited to passive transport/ release of molecules in the interior. Future work may focus on using non-canonical amino acids to impart functionalizable interior surfaces to allow orthogonal functionalization of the interior and exterior surfaces (ten Cate et al., 2006). 

The modified Mth RIRl, Mxe GyrA, and Ssp DnaB mini-inteins have been recently applied to the isolation of proteins with an N-terminal cysteine residues (29,30). These inteins undergo temperature- and pH-dependent C-termi-nal cleavage when the N-terminal cysteine residue of the intein is substituted with alanine (Table 2). The target protein is recombinantly expressed as a fusion protein with the C-terminal intein tag (31) (Fig. 3B). After intein splicing the protein that possesses N-terminal cysteine is generated. Moreover, such a protein can be obtained by total chemical synthesis and different chemical labels or non-canonical amino acids can be site-specifically incorporated into the sequence. 

The addition of a non-canonical amino acid to the genetic code requires - in the first case - additional components of the protein producing system a noncanonical amino acid, an exogenous tRNA/aminoacyl-tRNA synthetase pair, and an unique codon that specifies the amino acid of interest. Orthogonality between the exogenous translational components (Scheme 1-30) and their endogenous opposite numbers is the key feature of this approach. With the effect... 

Link, A. J., Mock, M. L., and Tirrell, D. A. Non-canonical amino acids in protein engineering. Current Opinion in Biotechnology 14, 603-609 (2003). 

Schoffelen S, Lambermon MHL, Eldijk MBV, Hest JCMV (2008) Site-specific modification of Candida antarctica lipase B via residue-specific incmporation of a non-canonical amino acid. Bioconjug Chem 19 1127-1131... 

Johnson JA, Lu YY, Van Deventer JA, Tirrell DA (2010) Residue-specific incorporation of non-canonical amino acids into proteins recent developments and applications. Curr Opin Chem Biol 14 774-780... 

Non-canonical Amino Acids as Probes of Biological Processes. 206... 

Over the past 15 years, we have made many variants of aECM proteins, including photocrosslinkable versions that contain the photosensitive non-canonical amino acid p-azidophenylalanine [27]. Our most recent experiments in this area are being done in collaboration with Teresa Ku and Arthur Riggs at City of Hope, and are directed toward the development of matrices for maturation and transplantation of human pancreatic p-ceUs for treatment of Type 1 diabetes [28]. 

Our studies of non-canonical amino acids were motivated initially by an interest in making proteins with novel properties. Our interest broadened when our colleague Daniela Dieterich suggested that pulsed metabolic labeling of cellular proteins with non-canonical amino acids might provide a method for time-resolved analysis of protein synthesis in neurons. With Daniela and Erin Schuman, we developed this idea into the BONCAT (bio-orthogonal non-canonical amino acid tagging) method shown in Scheme 2 [33]. 

In the BONCAT method, the cellular system of interest (cultured cells, tissue slices, or live animals) is pulse-labeled with a non-canonical amino acid that carries a reactive side chain. In our initial experiments, we used azidohomoalanine (Aha) as the label because Aha-Iabeled proteins can be selectively tagged with dyes or affinity reagents through copper-catalyzed or strain-promoted azide-alkyne cycloaddition reactions [34-36]. Tagged proteins can then be separated from other... 

S. Schoffelen, M.H.L. Lambermon, M.B. van Eldijk, J.C.M. van Hest, Site-Specific Modification of Candida antarctica Lipase B via Residue-Specific Incorporation of a Non-Canonical Amino Acid. Bioconjugate Chem., 19 (6) 1127-1131, 2008. 

An even more sophisticated approach comprises protein synthesis by cell-free expression as the amino acids added in a cell-free system are less exposed to bacterial metabolism, efficiency and selectivity of incorporation of specific isotope-labeled amino acids are only marginally hampered by isotope scrambling often observed in in vivo expression systems [19]. Even non-canonical amino acids can be incorporated allowing for site-specific labeling of proteins [20, 21]. 

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