Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

Less than a decade ago, the ability to generate proteins with unnatural modifications was a Herculean task available only to specialty labs. Recent advances make it possible to generate reasonable quantities of protein with un- natural amino acids both in vitro and in vivo . The combination of solid-phase peptide synthesis and enzymatic or che- moselective ligation now permits construction of entirely-synthetic proteins as large as 25 kD. Incorporation of recombi- nant fragments (expressed protein ligation) allows unnatural modifications near protein termini in proteins of virtually any size. Site-specific modification of small quantities of protein at any position can be achieved using chemically-acylated tRNA. Microelectroporation now extends this method to cells like mammalian neurons, and combination with RNA- display makes unnatural proteins compatible with combinatorial methods. Widespread or residue-speci fic methods of amino acid replacement are especially suitable for the production of biomaterials, and bacteria have been engineered to expand the repertoire of amino acids available for this technique. Excitingly, the wholesale addition of engineered tRNAs and synthetases to bacteria and yeast now makes site-specific incorporation of unnatural amino acids possible in living cells with no chemical intervention. Methods of expanding the genetic code at the nucleic acid level, including 4-base codons and unnatural base pairs, are becoming useful for the addition of multiple amino acids to the genetic code. These recent advances in unnatural protein engineering are reviewed with an eye toward future challenges, including methods of creating nonpeptidic molecules using templated synthesis.