MOLECULAR CHARACTERIZATION OF RECOMBINANT GREEN FLUORESCENT PROTEIN BY FLUORESCENCE CORRELATION MICROSCOPY

The cDNA for the green fluorescent protein (GFP) of Aequorea victoria has been expressed in transformed cells of Saccharomyces cerevisiae an d the recombinant GFP isolated. Protonation and deprotonation of the c loned and purified GFP produced major effects on its spectral absorpti on characteristics with an increase in pH enhancing the fluorescence e mission of the GFP more than twofold. Finally, molecular characterisat ion of GFP by fluorescence correlation microscopy in a minimal target volume of 1 fL yielded a translational diffusion coefficient (D-T) of 8.7x10(-7) cm(2).sec(-1), equivalent to a Stokes radius of 2.82nm for a monodisperse globular protein of 27kDa. (C) Academic Press, Inc.