Báo cáo khoa học: A new bright green-emitting fluorescent protein – engineered monomeric and dimeric

A new bright green-emitting fluorescent protein –

engineered monomeric and dimeric forms

Robielyn P. Ilagan1

, Elizabeth Rhoades1

, David F. Gruber2

, Hung-Teh Kao3

, Vincent A. Pieribone4

and Lynne Regan1,5

1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

2 Department of Natural Sciences, Baruch College and The Graduate Center, City University of New York, NY, USA

3 Department of Psychiatry and Human Behavior, Brown University, Providence, RI, USA

4 The John B. Pierce Laboratory, Yale University, New Haven, CT, USA

5 Department of Chemistry, Yale University, New Haven, CT, USA

Introduction

Fluorescent proteins (FPs) have become ubiquitous

tools in biological and biomedical research. Since the

cloning and exogenous expression of green fluorescent

protein (GFP) from the jellyfish Aequorea victoria,

researchers have sought new variants of this protein,

as well as of other FPs, with properties that are

well-suited for a particular application [1–3]. Extensive

mutagenesis has been performed on FPs to better

Keywords

detection marker; fluorescence correlation

spectroscopy; fluorescent protein;

oligomeric states; relative brightness

Correspondence

L. Regan Department of Molecular

Biophysics and Biochemistry, Yale

University, New Haven, CT 06520, USA

Fax: (203) 432 5175

Tel: (203) 432 9843

E-mail: [email protected]

Note

The nucleotide sequence data are available

in the DDBJ ⁄ EMBL ⁄ GenBank databases

under the accession number FN597286 and

the protein sequence data are in Uni￾ProtKB ⁄ TrEMBL with the accession number

D1J6P8.

(Received 22 December 2009, revised 5

February 2010, accepted 15 February 2010)

doi:10.1111/j.1742-4658.2010.07618.x

Fluorescent proteins have become essential tools in molecular and biologi￾cal applications. Here, we present a novel fluorescent protein isolated from

warm water coral, Cyphastrea microphthalma. The protein, which we

named vivid Verde fluorescent protein (VFP), matures readily at 37 C and

emits bright green light. Further characterizations revealed that VFP has a

tendency to form dimers. By creating a homology model of VFP, based on

the structure of the red fluorescent protein, DsRed, we were able to make

mutations that alter the protein’s oligomerization state. We present two

proteins, mVFP and mVFP1, that are both exclusively monomeric, and

one protein, dVFP, which is dimeric. We characterized the spectroscopic

properties of VFP and its variants in comparison with enhanced green fluo￾rescent protein (EGFP), a widely used variant of GFP. All the VFP vari￾ants are at least twice as bright as EGFP. Finally, we demonstrated the

effectiveness of the VFP variants in both in vitro and in vivo detection

applications.

Structured digital abstract

l MINT-7709188: VFP (uniprotkb:D1J6P8) and VFP (uniprotkb:D1J6P8) bind (MI:0407) by

classical fluorescence spectroscopy (MI:0017)

l MINT-7709201: VFP (uniprotkb:D1J6P8) and VFP (uniprotkb:D1J6P8) bind (MI:0407) by

fluorescence correlation spectroscopy (MI:0052)

l MINT-7709216, MINT-7709247, MINT-7709237: VFP (uniprotkb:D1J6P8) and VFP (uni￾protkb:D1J6P8) bind (MI:0407) by molecular sieving (MI:0071)

Abbreviations

dVFP, dimeric VFP; EC, extinction coefficient; EGFP, enhanced GFP; FCS, fluorescence correlation spectroscopy; FMRP, fragile X mental

retardation protein; FP, fluorescent protein; GFP, green fluorescent protein; GST, glutathione S-transferase; hpf, hours post-fertilization;

mVFP, monomeric VFP; QY, quantum yield; tD, diffusion time; t½, half time; T-Mod, TPR-based recognition module; TPR, tetratricopeptide

repeats; VFP, vivid Verde fluorescent protein.

FEBS Journal 277 (2010) 1967–1978 ª 2010 The Authors Journal compilation ª 2010 FEBS 1967