Incorporating Unnatural Amino Acids into Recombinant Proteins and Peptides

Unnatural amino acids offer exciting possibilities for expanding the functional repertoire of proteins and peptides. Here's how these non-canonical building blocks can be incorporated into recombinantly expressed proteins or peptides:

1. Expanded Genetic Code:

This elegant method introduces a specially designed tRNA/synthetase pair orthogonal to the host's existing system. The orthogonal tRNA recognizes the unnatural amino acid and the corresponding synthetase attaches it to the tRNA. During protein synthesis, this system allows the unnatural amino acid to be incorporated at specific codons, effectively expanding the genetic code.

2. Amber Suppression:

This strategy exploits the amber stop codon (UAG). An orthogonal tRNA/synthetase pair is engineered to recognize the amber codon and insert the unnatural amino acid instead of halting translation. By placing the amber codon at the desired position within the gene, the unnatural amino acid is incorporated site-specifically.

3. Chemical Modification:

This versatile approach involves producing the recombinant protein or peptide with a reactive group, typically a cysteine residue. A targeted chemical reaction then modifies the cysteine, introducing the desired unnatural amino acid. This method offers flexibility and is particularly useful for introducing modifications post-translationally.

4. Site-Directed Mutagenesis:

This classic method involves introducing specific mutations in the gene sequence to replace a natural amino acid with the desired unnatural amino acid. This is achieved through PCR using primers containing the desired mutation, resulting in a gene encoding the modified protein.

These methods empower researchers to incorporate unnatural amino acids into recombinant proteins and peptides, opening up a world of possibilities for engineering proteins with enhanced properties, novel functions, and therapeutic potential.