The incorporation of non-canonical amino acids into proteins enables us to alter the chemical and/or physical properties of proteins. In this work, we seek to develop high-affinity antibody single-chain variable fragments (scFvs) with altered chemical compositions by replacing the methionine residues in the fragments with one of several non-canonical amino acids that are close structural analogs of methionine. Two amino acids of interest to us, homopropargylglycine (Hpg) and azidohomoalanine (Aha), allow for the incorporation of chemical functionalities useful for future modification of antibody fragments using click chemistry techniques. A third amino acid of interest, norleucine (Nrl), lacks the easily-oxidizable thioether present in methionine side chains and may enable the development of proteins more resistant to oxidative damage. All of these amino acids can nearly quantitatively replace methionine under the appropriate expression conditions. Using a model anti-digoxigenin scFv displayed on the E. coli cell surface with an Lpp-OmpA' display anchor, we can estimate the display levels and dissociation rate constants of the scFvs using flow cytometry. Replacing methionine with any of the non-canonical amino acids described above results in decreased binding activity and/or display levels of the compositionally altered scFvs. We have used error-prone PCR and fluorescence-activated cell sorting (FACS) in an attempt to improve the properties of these proteins. After construction of a library based on the initial scFv construct (Antidig Lib 1), we screened the library for cells capable of increased binding to high amounts of digoxigenin after expression in medium containing Hpg. After three rounds of sorting, we constructed a second error-prone PCR library (Antidig Lib 2) based on the thrice-enriched population isolated from the first library. We screened Antidig Lib 2 using increasing levels of stringency under three different sets of expression conditions. Four rounds of sorting were completed after expression in media supplemented with each of the amino acids Hpg, Aha, and Nrl. Using kinetic competition methods, we have isolated clones possessing low apparent dissociation rate constants with altered compositions at the positions that normally contain methionine. Sequence analysis of clones isolated after three or more rounds of screening of Antidig Lib 2 expressed in Hpg-, Aha-, or Nrl-containing medium indicates that a number of commonly occurring mutations are shared amongst the various isolated populations despite the distinct amino acid contexts employed during the course of screening. Important differences in mutational frequencies between the populations are also observed. We are interested in the contributions of individual mutations to the affinity of the scFv for its cognate antigen and how these contributions change depending on the identity of the amino acids incorporated at positions normally occupied by methionine. Understanding these contributions may enable us to better understand fundamental aspects of protein folding and stability and how subtle changes in amino acid side chain identities affect these properties. From an engineering perspective, performing directed evolution experiments with proteins containing non-canonical amino acids may enable the development of therapeutically relevant proteins with properties that can only be introduced by incorporating non-canonical amino acids.