GFP, presented by figure 1A, is a 238 amino acid protein that adapts a beta barrel fold. This particular structure, containing a chromophore is responsible for its fluorescent properties and if the fold is altered the fluorescence is lost. Given the usefulness of GFP in various aspects of molecular biology, a number of different protocols for its expression and purification have been designed. It is a relatively easy and stable protein to purify, hence being treated as a way of learning techniques and methods used in modern protein cloning and obtaining recombinant proteins. GFP chosen for the following experiment has a monomeric structure and possesses one mutation. E.Coli is an organism chosen for the expression of the recombinant GFP. This bacteria is one of the most common choices for the recombinant proteins production since it has very high and effective growth kinetics, doubling its amount in just 20 minutes. The bacteria’s optimal growth is at 37 degrees Celsius in glucose and salt media, which is relatively cheap and easy to provide, making the E.Coli the appropriate organism suitable for the experiment.
Due to the use of polyhistidine-tags (His tags), the GFPs affinity for divalent metal ions, such as Ni2+ increases greatly which enables the efficient and effective use of Immobilized Metal Affinity Chromatography (IMAC) which is a convenient method of purification but cannot be implemented without the use of his tags.