Return to search

Expression and characterization of a novel orange flourescent protein cloned from the cnidarian tube anemone cerianthus sp. / CUHK electronic theses & dissertations collection

A crystal structure of OFP has been solved at 2.0 A resolution. It reveals that OFP is tetrameric and exists as a dimer of homo-dimer. It adopts the characteristic 11-stranded beta-can structure of GFP-like proteins. Based on the crystal structure, four special mutants were created primarily by site-directed random mutagenesis and error-prone PCR techniques. The first mutant is named Y37F in which a substitution of tyrosine (Y37) for phenylalanine creates a green emitter with an excitation maximum of 480 nm. The emission spectrum of Y37F is similar to that of EGFP and peaked at 498 nm. The second mutant is named K79R in which a substitution of lysine (K79) for arginine creates a red emitter with an excitation maximum of 548 nm. While the excitation maximum of K79R was literally unchanged with respected to OFP, the two emission maxima of K79R are both red-shifted to 512.5 nm and 583 nm. The third mutant is a dimeric mutant named OFP2 with W118K and V120E mutations. The absorption spectrum of OFP2 is identical to that of OFP. The emission spectrum of OFP2 has two emission maxima at 498 nm and 568 nm, respectively. Only bright orange fluorescence maximum at 568 nm is observed when OFP2 is excited at 515 nm or above. When OFP is excited between 430 nm and 470 nm, however, roughly equal amount of green emission maximum at 498 nm is also observed. The fluorescence quantum yield of OFP2 is about the same as OFP. The last mutant is named OFPm. It is a monomeric mutant of OFP2. A total of seven extra mutations (T141A/T144H/F155V/Y188P/K192S/R194S1N199A) were introduced on OFP2. The absorption maximum of OFPm is slightly red-shifted to 560 nm as compared to OFP2. The emission spectrum of OFPm has two emission maxima at 504 nm and 576.5 nm, respectively. Only red fluorescence maximum at 576.5 nm is observed when OFPm is excited at 525 nm or above. When OFPm is excited between 430 nm and 470 nm, however, the emission is mainly green fluorescence peaked at 504 nm with small amount red light peaked at 576.5 nm. The fluorescence quantum yield of OFPm is about 0.32. / A novel Orange Fluorescent Protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. This protein consists of 222 amino acid residues with the 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 (egFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-Orange fluorescent protein (KO), and a mere 21% identity to Aequorea victoria green fluorescent protein (GFP). Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 483 and 515 nm. Bright orange fluorescence maximum at 568 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 500 nm, a considerable amount of green emission maximum at 498 nm was also observed. It has a fluorescence quantum yield (phiF) of 0.64 at 25°C. The molar absorption coefficients (epsilon) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M-1 · cm -1, respectively. Its fluorescent brightness (epsilon · phi F) at 25°C is 38,400 M-1 · cm -1. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12. OFP was readily expressed as soluble protein in Escherichia coli at 37°C. / Ip, Tsz Ming. / "November 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4733. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 139-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344138
Date January 2007
ContributorsIp, Tsz Ming., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, theses
Formatelectronic resource, microform, microfiche, 1 online resource (xix, 149 p. : ill.)
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Page generated in 0.009 seconds