| The luminescent system ofAequorea consisted of photoprotein-aequorin and green fluorescent protein (GFP). GFP As a good in vivo marker and aequorin as molecular marker and Ca2+ indicator, had been widely applied in many research areas in biology. Studies of the bioluminescent mechanisms of jellyfish to date have focused primarily one species, Aequorea victoria. The present paper describes the luminescent system of other species of Aequorea found on the coastal waters of Xiamen, P.R.CHINA.Two new aequorin genes - aeqxm and aeqxxm -were isolated from fellyfish Aequorea macrodactyla and Aequorea parva respectively, which are commonly found in the warmer waters on the coastal region of the East China Sea. The DNA sequences of the two genes have no introns and each one contains an ORF of 585 bp in full-length encoding a 195-aa protein. The two genes of aeqxm and aeqxxm share nucleotide homologies of 80.7% and 85.1 % with AEVAQ440X respectively, and the corresponding proteins share amino acid homologies of 84.7% and 84.2% with AEVAQ440X. High amino acid homolosfy (94.4%) was found between apoaeqxm and apoaeqxxm. The two genes were cloned into expression vector pTO-T7 respectively, and the expression yields were amounted to 40% of the total protein in E.coli BL21. The activities of the two photoproteins were reconstituted by incubating the expressed apoproteins with coelenterazine f. In the presence of Ca ion, both of the regenerated aeqxm and aeqxxm exhibited an emission peak at the wave length of 470nm. The expressed apoproteins have normal bioactivities.A new green fluorescent protein gene gfpxm was isolated from Jellyfish Aequorea macrodactyla by the same method of combination of PCR amplification and Southern hybridization analysis. The gfpxm gene containsthree extrons and two introns spread over 1042bp of genomic DNA, the entire coding region of cDNA is 717nt identical to the wild-type gfp AeqgfplO cDNA in length. The amino acid sequence of GFPxm deduced from the nucleotide sequence is 238-aa-residue, sharing homology of 83. 6% with that of AeqGFPlO. The entire coding sequence was cloned into the pTO-T7 expression vector and expressed in E coli BL21. The expression yield of GFPxm was amounted to 50% of the total protein. Compared with GFP of A. victoria, the expressed GFPxm exhibited an excitation peak at a higher wave length of 476nm and an emission peak at a lower wave length 496nm with a higher quantum yield of 1.0. The fluorescent of GFPxm is significant stable, showing strong resistant to heat, alkaline, denarurants and salts.However, the drawback of only maturing to fluorescence at low temperature limited its applications. Twelve mutants of GFPxm were obtained by further site-directed mutagenesis and DNA shuffling.Two mutants, i.e, GFPxm 18 and GFPxm 19 produced enhanced fluorescence when expressed in E.coli at higher temperature (37癈). The two sets of substitutions caused a very small shift of excitation and emission maximum. GFPxm 16, ShG24 and GFPxm 161 had small red-shift of excitation-emission maxima up to 485-500 nm and 506-511 nm.Three mutants GFPxm 162, GFPxm 163 and OFPxm with further red-shifted spectrum were obtained. The longest emission maxima of 525 nm was obtained when th?T in position 203 of GFPxml6 was replaced by F. The same T203Y substitution of GFPxm 16 and ShG24 provided similar excitation-emission spectra. GFPxm 163 had excitation peak of 512nm and emission peak of 523nm, fluorescence yellowish green. GFPxm 163 had the strongest fluorescence intensity in GFPxm mutants. OFPxm had an excitation peak at 509 nm and an emission peak at 523 nm which is yellowish green, but the protein is orange observed by eyes. The mutant could reach high expression level and matured at higher temperatu?e but the fluorescence intensity was comparatively low owing to low quantum yield.Other three mutants with two emission peak were obtained by site-directedmutagenesis. The mutant GFPxml91uv had a major excitation peak at 498 nm that was about three times of the minor peak at 3...
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