Protein engineering with unnatural amino acids

•Protein engineering with uAAs is a valuable technique with applications in the study of protein function, protein modification and biophysics.•The unique chemistry of some uAAs can allow otherwise impossible experiments to be performed.•The ability to improve protein function with unnatural amino acids has lagged behind their other applications.•Recent technical breakthroughs will enhance our ability to improve protein function using unnatural amino acids in the future.

Protein engineering has become an extensively used tool in many fields, allowing us to probe protein function, characterize proteins using a range of biophysical techniques, chemically modify proteins and improve protein function for medical and industrial applications. It is now possible to site-specifically incorporate unnatural, or non-canonical, amino acids (uAAs) into proteins, which has had a major impact on protein engineering. In this review, we discuss the recent technical developments in the field and how uAA–protein engineering is becoming an increasingly valuable molecular tool, with the unique chemical functionalities of some uAAs allowing a range of otherwise impossible experiments to be performed. Finally, the impediments that have resulted in a relatively small number of recent studies in which uAA–protein engineering has been used to improve protein function are discussed, alongside some of the recent technical developments that may serve to overcome these obstacles.