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Marine Biotechnology

, Volume 9, Issue 4, pp 469–478 | Cite as

Characterization of Novel Orange Fluorescent Protein Cloned from Cnidarian Tube Anemone Cerianthus sp.

  • Denis Tsz-Ming Ip
  • Kam-Bo Wong
  • David Chi-Cheong Wan
Original Article

Abstract

A novel orange fluorescent protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. It consists of 222 amino acid residues with a calculated molecular mass of 25.1 kDa. A BLAST protein sequence homology search revealed that native OFP has 81% sequence identity to Cerianthus membranaceus green fluorescent protein (cmFP512), 38% identity to Entacmaea quadricolor red fluorescent protein (eqFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-orange fluorescent protein (KO), and a mere 21% identity to green fluorescent protein (GFP). It is most likely that OFP also adopts the 11-strand β-barrel structure of fluorescent proteins. Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 487 and 513 nm. A bright orange fluorescence maximum at 573 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 515 nm, a considerable amount of green emission maximum at 513 nm was also observed. It has a fluorescence quantum yield (Φ) of 0.64 at 25°C. The molar absorption coefficients (ɛ) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M-1−1 • cm-1−1, respectively. Its fluorescent brightness (ɛ Φ) at 25°C is 38,400 M−1-1 • cm−1-1. Like other orange-red fluorescent proteins, OFP is also tetrameric. It was readily expressed as soluble protein in Escherichia coli at 37°C, and no aggregate was observed in transfected HeLa cells under our experimental conditions. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12.

Keywords

Cerianthus cnidarian green fluorescent protein orange fluorescent protein 

Notes

Acknowledgments

This research was supported by RGC Earmarked Grant CUHK4571/05M and Direct Grant from CUHK.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Denis Tsz-Ming Ip
    • 1
  • Kam-Bo Wong
    • 1
  • David Chi-Cheong Wan
    • 1
  1. 1.Department of BiochemistryThe Chinese University of Hong KongShatin, New TerritoriesChina

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