A unique red fluorescent protein of silkworm bearing two photochromic moieties

Abstract

A silkworm excretory red fluorescent protein (SE-RFP) having light-dependent activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified. Light was observed to be essential also for the SE-RFP synthesis as it was produced only when silkworms were reared in light. SE-RFP has exhibited a high fluorescence quantum yield of 0.86. The apparent mass of native SE-RFP was about 1100 kDa as analysed by gel filtration chromatography. Two photochromic moieties associated with the SE-RFP, namely tetrapyrrole-I (TP-I) and tetrapyrrole-II (TP-II), were isolated by employing TLC and HPTLC techniques. The purified tetrapyrroles were characterized by UV-absorption, fluorescence, atomic absorption and FT-IR spectral analyses. The molecular masses of TP-I and TP-II were 535 and 870 Da, respectively, as determined by ESI-MS and MALDI-TOF-MS. The molar ratio of TP-I to TP-II was 1.14 : 1.00, and a total of 7.251 micromol tetrapyrroles (TP-I + TP-II) were found to be present per mg of SE-RFP. TP-I and TP-II were identified as chlorophyll derivatives, namely, pyropheophorbide a and pheophytin a, respectively. Hence, the SE-RFP was concluded to be a unique insect red fluorescent protein having two photochromic moieties and potent photobiological activity.