Protein engineering has become an extensively used tool in many fields which allow us to study protein functions and characterize proteins using range of available biophysical methods. With the genetic code expansion, it is now possible to incorporate functional groups and other properties in the proteins which are naturally not present. Site specific incorporation of unnatural amino acid (uAA) is widely used in protein engineering. However, incorporation of multiple uAAs remains a significant challenge. Tunability of protein expression for multiple uAAs incorporation using orthogonal ribosome remains another important challenge.