Green Fluorescent Protein Purification
The GFP purification activity is designed to following the pGLO Bacterial Transformation kit. After the transformation process, students begins with removing the transformed E.Coli bacteria from agar plates and allows to multiply in liquid nutrient media. Then the bacterial host are lysed using specific enzyme thus allowing GFP to be released. Finally, debris of the bacteria including other proteins, chromosomal DNA are filtered out through a specific technique called "chromatography." The unique fluorescent properties of GFP enables the entire process to be observed through long-wave UV light.
* Cultivate the transformed E.Coli bacteria from agar plates and removes multiply in liquid nutrient media.
*Glowing pellet proves the existance of Green Fluorescent Protein in E.Coli host
*Lysozyme
Lysozyme is an enzyme that functions to degrade (or lyse) the bacterial cell wall, by cleaving polysaccharide (sugar) residues in the cell walls. The subsequent freeze-thaw step used in this lesson aids in the complete disruption of the wall and internal membrane. Complete disruption or "lysis" releases soluble components, including GFP. Lysozyme is naturally found in human tears, acting as a bactericidal agent to help prevent bacterial eye infections. Lysozyme gets its name from its natural ability to "lyse" bacteria.
*Lysozyme is added into the the culture; Along with freeze-thaw method, the membrane of E.Coli cell membrane is broken down.
*The mixture of GFP and cell debris is centrifuged again. This time only the supernatant contains GFP.
GFP contains an abundance of hydrophobic amino acids. Salt solution causes the three-dimensional structure of proteins to actually change so that the hydrophobic regions of the protein move to the exterior of the protein and the hydrophilic ("water-loving") regions move to the interior of the protein. At this stage, the supernatant will fluoresce bright green upon exposure to UV light. The chromatography column is developed by Bio-rad company which contains a matrix of microscopic hydrophobic beads. When your sample is loaded onto this matrix in very salty buffer, the hydrophobic proteins should stick to the beads. The more hydrophobic the proteins, the tighter they will stick.
*"Equilibrium buffers", "Binding buffers", "TE buffers" are respectively added into the column. The last one, TE buffer, with the lowest salt concentration, changes the 3D shape, and turns the hydrophilic side out.
*The filtrate are a mixture of highly condensed of water and Green Fluorescent Protein.
*Comparing to the filtrate with water (control group)