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GFP Protein expression, purification and analysis.

Green Fluorescent Protein - Bioluminescence is the ability of an organism to produce color and light. The term comes from the Greek “ bios” living and the Latin “lumen” light. It is estimated that ninety percent of deep sea marine organisms can bioluminesce, whereas terrestrial bioluminescent organisms are less common. The two best known land bioluminators are fireflies and New Zealand glow worms. In bioluminescent jellyfish, color and light are observed when light energy is transferred to the green fluorescent protein. This results in a change in the conformation (shape) of the protein, producing the light and color. In recent years, Green Fluorescent Protein has been isolated and engineered for laboratory use.

Green Florescent Protein (GFP) Purification.

Bacteria cells that have been transformed with the pGLO plasmid and are found to express GFP can now be used to produce and purify the protein. To separate the GFP from the other endogenous proteins in the bacteria, hydrophobic Interaction Chromatography (HIC) is employed. GFP is soluble but contains several stretches of hydrophobic amino acids. In the presence of high salt buffer the three dimensional structure of the protein changes such that the hydrophobic regions of the protein are exposed and the hydrophilic regions are shielded. These hydrophobic amino acids tend to stick to other hydrophobic substances, ie to the hydrophobic beads of the HIC column. When salt is removed, the 3D structure of the protein will return to normal exposing the hydrophilic regions, the protein will no longer stick to the column and is eluted from the column.

Four different buffers will be used in the HIC procedure:

Equilibriation buffer: A medium salt buffer (2M ammonium sulfate) is used to ‘prime’ the column for binding GFP.
Binding buffer: Binding buffer (4M ammonium sulfate) is added to the bacterial cell lysate causing the hydrophobic regions of the GFP to be exposed.
Wash buffer: A medium salt buffer (1.3M ammonium sulfate) is used to wash weakly associated proteins from the column.
Elution buffer: A low salt buffer (TE:10mM Tris/EDTA) is used to wash GFP from the column.

To determine protein size, level of purity and yield, SDS-PAGE will be performed. For detailed information on this method please refer to the background section of the lab ‘Determining the molecular weight of lysozyme’.

Coomassie blue will be used to visualize proteins on the gel. Coomassie dyes (also known as Coomassie Brilliant Dyes) are a family of dyes commonly used to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel electrophoresis (SDS-PAGE and BN-PAGE, respectively) gels. The gels are soaked in dye and excess stain is then eluted with a solvent ("destaining"). This treatment allows for visualization of protein bands. The gel usually contains a set of molecular weight marker (proteins of pre-determined weight) so that protein molecular weight can be estimated in an unknown solution during the visualization. The neutral ionic species of coomassie dye binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with arginine, the aromatic amino acids, and histidine. (Ioannis, P et al A. Analytical and Bioanalytical Chemistry, Volume 391, Number 1, May 2008 , pp. 391-403(13)

Transformation Efficiencies - Transformation efficiencies are a way to determine how many cells were transformed per ug of plasmid DNA used. The colonies we count after the plates have incubated overnight originally grew from one transformed cell, called a transformant (provided streaking out of the plates was done properly). The calculation for transformation efficiency is:

Transformation Efficiency Equation

(In the laboratory we will use 0.05ug of pGLO, our final volume will be 0.50ml, and we will plate a volume of 0.20ml)

(An example of 40 colonies using the laboratory conditions and volumes would produce 2000 transformants/ug of DNA)

In research laboratories the transformation efficiencies will usually be between 1x104 to 1x107 transformants per ug of DNA. Transformations are never 100% efficient.

 

REF: Edvotek for MdBioLab by Jennie Queen-Baker

Biochemistry Laboratory by Robert Boyer