Characterization of recombinant photoconverting green fluorescent Akanes

Abstract

Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.

Keywords: Akane; Scleronephthya gracillima.Abbreviations: DTT, dithiothreitol; EtOH, ethanol; GFPs, green fluorescent proteins; MeCN, acetonitrile; Mw, molecular weight; SDS, sodium dodecyl sulphate; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; SRM, super resolution microscopy; TFA, trifluoroacetic acid; green fluorescent protein; multicolour fluorescent protein; photoconversion.