Creating a Streptavidin-meGFP Protein Construct
Mentor 1
Ionel Popa
Start Date
28-4-2023 12:00 AM
Description
Streptavidin [alive] (SAA) is a homo-tetramer protein with high affinity for biotin. Biotin is a cofactor necessary for metabolic enzymes such as carboxylases and decarboxylases, which produce metabolites in gluconeogenesis, lipogenesis, and amino acid metabolism. Streptavidin-biotin binding is one of the strongest noncovalent interactions found in nature, far beyond the strength of other protein-ligand binding. One streptavidin molecule can bind four biotin molecules, amplifying weak signals and improving detection sensitivity. The interaction is resistant to many disruptive denaturing conditions, including organic solvents, detergents, and extreme temperature or pH, underlying its importance in the field of molecular biology. Engineering biotin sites on proteins such green fluorescent protein (GFP) allows for its specific tagging and visualization. In this project, a meGFPSAA construct was created using restriction digestion on an initial pQEmeGFP construct to remove the meGFP fragment, which was ligated into a digested pQTEVSAA vector to create the final construct, pQTEVmeGFPSAA. This was expressed in XL-1 blue cells and confirmed via gel electrophoresis and next-generation sequencing. The meGPFSAA fragment will be transferred to a pQE vector, containing biotin sites at either end, for improved expression in protein purification. This construct will be used to perform initial binding experiments between the fluorescent protein with its substrate, streptavidin. Creating a single plasmid coding for the streptavidin and meGFP proteins increases the efficiency of both their expression and interactions with one another. The streptavidin-biotin interaction can be used to attach biomolecules to one another or to a support, aiding in purifying and detecting biomolecules. In the Popa Laboratory, this interaction will be used in binding experiments with protein hydrogels, polymeric materials built by cross-linked protein monomers. Protein hydrogels can be utilized to understand the mechanical unfolding and subsequent refolding of proteins via rheometry by applying tensile force and measuring its effect.
Creating a Streptavidin-meGFP Protein Construct
Streptavidin [alive] (SAA) is a homo-tetramer protein with high affinity for biotin. Biotin is a cofactor necessary for metabolic enzymes such as carboxylases and decarboxylases, which produce metabolites in gluconeogenesis, lipogenesis, and amino acid metabolism. Streptavidin-biotin binding is one of the strongest noncovalent interactions found in nature, far beyond the strength of other protein-ligand binding. One streptavidin molecule can bind four biotin molecules, amplifying weak signals and improving detection sensitivity. The interaction is resistant to many disruptive denaturing conditions, including organic solvents, detergents, and extreme temperature or pH, underlying its importance in the field of molecular biology. Engineering biotin sites on proteins such green fluorescent protein (GFP) allows for its specific tagging and visualization. In this project, a meGFPSAA construct was created using restriction digestion on an initial pQEmeGFP construct to remove the meGFP fragment, which was ligated into a digested pQTEVSAA vector to create the final construct, pQTEVmeGFPSAA. This was expressed in XL-1 blue cells and confirmed via gel electrophoresis and next-generation sequencing. The meGPFSAA fragment will be transferred to a pQE vector, containing biotin sites at either end, for improved expression in protein purification. This construct will be used to perform initial binding experiments between the fluorescent protein with its substrate, streptavidin. Creating a single plasmid coding for the streptavidin and meGFP proteins increases the efficiency of both their expression and interactions with one another. The streptavidin-biotin interaction can be used to attach biomolecules to one another or to a support, aiding in purifying and detecting biomolecules. In the Popa Laboratory, this interaction will be used in binding experiments with protein hydrogels, polymeric materials built by cross-linked protein monomers. Protein hydrogels can be utilized to understand the mechanical unfolding and subsequent refolding of proteins via rheometry by applying tensile force and measuring its effect.
