Proteins containing non-canonical amino acids can possess unique physical or chemical properties because of their novel compositions. Previous work has demonstrated that residue-specific replacement of a canonical amino acid in a protein with a non-canonical residue can be achieved with a wide variety of amino acid analogs. However, proteins synthesized in residue-specific replacement experiments often suffer loss of function. In this work, we seek to develop antibody fragments capable of binding antigens after replacement of methionine with a non-canonical amino acid. Using the Lpp-OmpA' E. coli cell surface display system, we have shown that an Lpp-OmpA'-anti-digoxin single-chain variable fragment (scFv) fusion protein can be expressed in the presence of several non-canonical amino acids. Interestingly, the level of protein expression varies with the non-canonical amino acid analog used and with the expression system employed. For example, the expression levels from cells induced in synthetic media in which methionine has been replaced with homopropargylglycine (Hpg) or azidohomoalanine (Aha) are nearly equal to the expression levels in cells in which the media were supplemented with methionine when an isopropyl-beta-D-thiogalactopyranoside- (IPTG) inducible expression system was utilized. Despite the high levels of protein synthesis observed in the IPTG-inducible system, flow cytometry experiments have revealed that most of the binding activity of the scFv is lost upon replacement of methionine with either Hpg or Aha. We are currently screening libraries containing mutagenized Lpp-OmpA'-scFv genes expressed in the presence of Hpg with the objective of isolating clones showing improved binding properties. When the media conditions described above are used with the arabinose-inducible pBAD expression system, poor levels of Lpp-OmpA'-scFv expression are observed in cells induced in media containing analogs of methionine. We attribute the low expression levels to the autocatalytic properties of the pBAD expression system. We are attempting to develop host strains that are better suited for expression of proteins containing non-canonical amino acids from an arabinose-inducible promoter.