A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis.
A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR. The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 beta-strands of hmGFP were similar to those of GFP from other species. The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification. The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized. The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm. The expression of hmGFP and the chimera IPL through hmGFP in CHO cells has shown that the fusion protein IPL through hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein.
Tu
Hongbin
H
Department of Biochemistry, The Open Laboratory for Marine Functional Genomics of State High-Tech Development, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, 510275, P.R., Guangzhou, China.
Xiong
Qian
Q
Zhen
Suilan
S
Zhong
Xiaofen
X
Peng
Lisheng
L
Chen
Huiping
H
Jiang
Xiaoyu
X
Liu
Wenhua
W
Yang
Wenli
W
Wei
Jianwen
J
Dong
Meiling
M
Wu
Wenyan
W
Xu
Anlong
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D049452:Green Fluorescent Proteins; D037402:Hydrozoa / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012615:Sea Anemones / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins