 Streptavidin is used in research, biotechnology, and medicine to form a non-covalent complex with a biotinylated ligand. For some applications where high affinity interaction is necessary, wild-type Streptavidin is an appropriate molecule to use. But in other situations, the use of wild-type Streptavidin can create a problem. Streptavidin is a homotetramer and has four high affinity binding sites. As a result, it combines multiple ligands and thus cross-linked them. This can be a problem when labeling molecules for imaging and tracking studies. As a tetramer, it may also be difficult to recombinantly fuse it to other molecules. We want it to create a monomeric Streptavidin that doesn't require oligomerization for function. Others have tried to engineer monomeric Streptavidin by introducing surface mutations. But in all cases, the engineered molecule had poor biophysical properties, including limited stability and affinity. In this work, we address these issues by designing a structural monomer that is stable and binds biotin with higher affinity. Such a molecule should be useful for various applications. Next, I would like to walk you through the process of how we engineered and characterized the molecule. The binding pocket of Streptavidin consists of residues from multiple subunits, including a tryptophan that forms a hydrophobic lid over bound biotin and is missing in the monomer binding site. However, there are Streptavidin homologs that form natural structural dimers, yet bind biotin with high affinity. Because their binding sites consist of residues from a single subunit, it may be possible to decouple biotin interaction from subunit association. We used homology modeling to build a monomer based on Streptavidin and Resavidin. To emulate the interaction between Resavidin and Biotin, we grafted the binding loop from Resavidin to Streptavidin and mutated other residues that may be involved in biotin binding. We also replaced the loop residues located at the opposite end of the beta barrel. Shortening the loop should increase the stability of the molecule without affecting its function. Dissociating Streptavidin exposes a lot of residues that were previously buried at the interface. These were mutated in the monomer to be consistent with their increased solvent exposure. The resulting molecule had comparable sequence identity to Streptavidin and Resavidin, indicating that it is a true hybrid between the two parent molecules. The desired molecule was experimentally tested. Our study shows that it is possible to engineer a stable and high-intensity monomeric streptavidin, which have useful properties that are acting in wild petriptavidin and other currently available streptavidin units. We first purified the protein from bacteria using affinity chromatography. The protein was then analyzed by circular dichroism and fluorescence polarization spectroscopy to obtain the denaturation temperature of 60 degrees and the dissociation constant of 2.8 nanomolar. We also fused the monomer to GFP to create a bifunctional molecule that is fluorescent and binds biotin. We demonstrated that it can bind a biotinylated cell surface receptor without aggregating it. The designed monomer can be used as a genetic fusion tag. For instance, it can be fused to a cell surface receptor for labeling with a biotinylated fluorophore. On the other hand, wild type streptavidin tetramer can be easily fused to a heterologous protein in a functional form. I hope this has been an informative session. Please go and check out the article which has been published in Biotechnology and Bioengineering. I can be reached at the number and email provided with the paper. I welcome any suggestions you may have on the design and the usage of the molecule. Thank you for watching.