CHEMICAL SYNTHESIS OF THE PRECURSOR MOLECULE OF THE AEQUOREA GREEN FLUORESCENT PROTEIN, SUBSEQUENT FOLDING, AND DEVELOPMENT OF FLUORESCENCE

The present paper describes the total chemical synthesis of the precur sor molecule of the Aequorea green fluorescent protein (GFP), The mole cule is made up of 238 amino acid residues in a single polypeptide cha in and is nonfluorescent, To carry out the synthesis, a procedure, fir st described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effec tiveness of the procedure has been demonstrated by the synthesis of va rious biologically active peptides and small proteins, such as human a ngiogenin, a 123-residue protein analogue of ribonuclease A, human mid kine, a 121-residue protein, and pleiotrophin, a 136-residue protein a nalogue of midkine, The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue pepti de was treated with anhydrous hydrogen fluoride to obtain the precurso r molecule of GFP containing two Cys (acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0 .1 M Tris HCl buffer (pH 8.0) in the presence of DTT, After several ho urs at room temperature, the solution began to emit a green fluorescen ce (lambda(max) = 509 nm) under near-UV light, Both fluorescence excit ation and fluorescence emission spectra were measured and were found t o have the same shape and maxima as those reported for native GFP, The present results demonstrate the utility of the segment condensation p rocedure in synthesizing large protein molecules such as GFP, The resu lt also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.