GFP are used as reporter gene, reporter gene is used to allow researchers to see if the gene of interest is expressed in an organism. GFP is a good reporter gene because it glows green fluorescence under UV lights when it is attached to the DNA of the gene of interest, reporting very clearly if gene is active in the organism. GFP is composed of 238 amino acid residues, in the center of GFP a single chromophore made of a single chain of four amino acids that is responsible for fluorescence colour without needing any cofactors. Because chromophore is well protected inside the cylindroid shape, therefore GFP can fluoresce in variety environments making it a highly versatile protein.
The appropriate site of positioning of GFP during protein fusion is at the primary sequence of the protein of interest, domains are tried to be avoided. GFP can be attached to both N terminal or C terminal of protein of interest, but typically one end of the protein contains the function domain that could be sterically hindered by addition of GFP, therefore GFP will be attach to the opposite end. If a protein contains signal sequence at the N terminal, and a retention sequence at C terminal, GFP will be placed immediately after the signal sequence and before the retention sequence, therefore it will be less likely to interfere with the function of either sequence. If the protein of interest is transmembrane protein, GFP cannot be attached near the transmembrane domain.
GFP displays two absorptions maxima at neutral form 397nm and anionic form 477 nm, blue to ultraviolet range, the emission is peak at 509 nm, light green range. Many different mutants of GFP have been engineered due to the evolving needs of researchers; in particular, blue fluorescent protein (BFP), cyan fluorescent protein (CFP), and yellow fluorescent protein derivatives (YFP).