Date of Award


Document Type



Santa Clara University

First Advisor

Zhiwen Zhang


Firefly luciferase is a bioluminescent protein commonly used as a bioluminescent tag in biological studies and applications. However, because the protein is fairly large in size, it is sometimes larger than the molecules it is intended to measure and is therefore not a sufficient tag in smaller applications. The active site of firely luciferase is also not well understood, making it difficult to engineer the protein without affecting its bioluminescent activity. In this paper, we discuss the experimental methods of Reverse Protein Engineering: a bioengineering technology that reduces the size of a protein while retaining its original function. This involves subcloning a core section of the protein, attaching a DNA library to the core to achieve a large pool of randomized peptide variants, and screening those variants for any bioluminescent properties. Successful conduction of this technique would achieve two goals: 1) create a peptide alternative to resolve the protein’s current size limitations and 2) confirm the importance of specific amino acids that might contribute to the active site’s activity. Our experiments show that Reverse Protein Engineering can be done to decrease the size of Firefly Luciferase (550 amino acids) to a much smaller peptidic version of the protein (less than 80 amino acids). However, to determine successful function of the peptide variants, more research in screening the peptides for bioluminescent activity needs to be done. In addition, Reverse Protein Engineering with a different range of amino acids within the core could further the chances of achieving a successful bioluminescent peptide variant of firefly luciferase.