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One stone, two birds: The Goal of the protein engineers and chemical biologist is to expand the building blocks of protein translation of eukaryotes and prokaryotes with amino acid surrogates (Unnatural amino acid). Currently, a number of unnatural amino acid was incorporated into protein with the help the methodology such as Expanding (sense codon reassignment or residue-specific incorporation) and engineering the genetic code (non-sense suppression or site-specific incorporation). These approaches can be applied to incorporate any desired functional group into protein which provides the protein with novel function properties such as bio-tagging, probing, enhanced functional and spectral properties which is inherited from the incorporated functional group. Despite the potential significance, there remain pitfalls such that these methods can incorporate only one functionality into the protein. Simultaneously, incorporating two different functionalities will provide the protein with a two different functions. One of the major significance is that it will open a new door in the peptidomimetics to synthesis novel antimicrobial peptides with different combination UAAs. To attain the challenging goal, here we have developed a simple, efficient and robust method for the incorporation of multiple unnatural amino acids (MUAA) in a single protein by coupling the above mentioned approaches. In this study, to prove the approach, we used GFP as model protein in which the methionine will be replaced globally with its analog L-homopropargylglycine through sense codon reassignment approach. Simultaneously, tyrosine analog 3,4-dihydroxy-L-phenylalanine (L-Dopa) will be incorporated in response to the non sense codon (Amber codon) inserted at Y66 (or) K15 with the help of an evolved Methanococcus jannaschii tRNA/synthetase pairs (mutant TyrRS). Now, the multi-functional protein has the potential ability for bio-orthogonal conjugation derived from the incorporated DOPA and L-Hpg can be chemoselectively modified with specific alkyne bearing reagent by means of a copper mediated azide-alkyne cycloaddition. This is the first study to demonstrate in vivo incorporation of MUAA into a recombinant protein through combination of two different methodologies. This combination will offer an extraordinary opportunity for protein engineers to create protein with novel functionality which expand the proteome of the cell. This approach will offer protein engineers to provide toolkits with unprecedented benefits and opportunities in the coming age of synthetic biotechnology.