Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(12):e28674.
doi: 10.1371/journal.pone.0028674. Epub 2011 Dec 8.

An Enhanced Monomeric Blue Fluorescent Protein With the High Chemical Stability of the Chromophore

Free PMC article

An Enhanced Monomeric Blue Fluorescent Protein With the High Chemical Stability of the Chromophore

Oksana M Subach et al. PLoS One. .
Free PMC article


Commonly used monomeric blue fluorescent proteins suffer from moderate brightness. The brightest of them, mTagBFP, has a notably low chemical stability over time. Prolonged incubation of mTagBFP leads to its transition from a blue fluorescent state with absorbance at 401 nm to a non-fluorescent state with absorbance at 330 nm. Here, we have determined the chemical structure of the degraded product of the blue mTagBFP-like chromophore. On the basis of mTagBFP we have developed an improved variant, named mTagBFP2. mTagBFP2 exhibits 2-fold greater chemical stability and substantially higher brightness in live cells than mTagBFP. mTagBFP2 is also 1.2-fold and 1.7-fold more photostable than mTagBFP in widefield and confocal microscopy setups, respectively. mTagBFP2 maintains all other beneficial properties of the parental mTagBFP including the high pH stability and fast chromophore formation. The enhanced photostability and chromophore chemical stability of mTagBFP2 make it a superior protein tag. mTagBFP2 performs well in the numerous protein fusions and surpasses mTagBFP as a donor in Förster resonance energy transfer with several green fluorescent protein acceptors.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Photochemical and biochemical properties of the purified mTagBFP2 protein.
(A) Time dependence of fluorescence for mTagBFP2, mTagBFP and EBFP2 in PBS, pH 7.4 at 37°C. (B) Excitation (dashed line), absorbance and emission (solid lines) spectra of mTagBFP2. (C) Photobleaching curves for purified mTagBFP2 (squares) and mTagBFP (circles) under epifluorescence illumination using metal halide arc lamp. According to the Student's t-test a difference between the photobleaching curves is statistically significant. (D) Dependence of fluorescence half-life times on pH for mTagBFP2 and mTagBFP at 37°C. (E) Time dependence of fluorescence for mTagBFP2 and mTagBFP denatured in 6 M guanidinium hydrochloride at 25°C. Error bars, s.d. (n = 3 (A, D, E); and n = 10 (C)).
Figure 2
Figure 2. Autocatalytic degradation of the TagBFP-like chromophore.
(A) Absorbance spectra of mTagBFP2 (blue) and mTagBFP (black) before and after incubation in 100 mM NaH2PO4, 300 mM NaCl, pH 8.0 at 37°C for 200 h. (B) Time dependence of absorbance for mTagBFP2 (blue) and mTagBFP (black) at 330 nm (dashed lines) and 401 nm (solid lines). Purified proteins were incubated in 100 mM NaH2PO4, 300 mM NaCl, pH 9.0 at 37°C. (C) SDS-PAGE analysis of mTagBFP2 and mTagBFP after incubation at 4°C (lanes 2 and 3) or at 37°C (lanes 4 and 5) for 770 h. M: molecular weight protein markers. Bands 1 and 2 show the total polypeptide chain and the polypeptide chain after cleavage inside the chromophore, respectively. (D) The ratio of the band 1 intensity to the band 2 intensity for SDS-PAGE analysis presented in Figure 2C. (E) Deconvoluted FT-ICR mass spectrum: isotopic distribution of the region corresponding to the band 2 (C). The deconvoluted average mass (19163.83 Da) is indicated by the red arrow. (F) Chemical scheme of hydrolysis of the mTagBFP-like chromophore. Error bars, s.d. (n = 2 (B); and n = 3 (D)).
Figure 3
Figure 3. Properties of mTagBFP2 in live mammalian cells.
Photobleaching curves for mTagBFP2 (squares) and mTagBFP (circles) expressed in HeLa cells under metal halide arc illumination (A) or 405 nm laser scanning confocal microscope illumination (B). Data represent an average of 32–65 cells per each protein. According to the Student's t-test a difference between the photobleaching curves is statistically significant. (C) Mean fluorescence brightness of HeLa cells expressing mTagBFP2 (solid line) or mTagBFP (dashed line). (D) Mean fluorescence of mTagBFP2- and mTagBFP-expressing HeLa cells corresponding to panel (C). Error bars, s.d. (n = 65 (A); n = 32 (B); and n = 3 (C)).
Figure 4
Figure 4. Fluorescence imaging of mTagBFP2 fusion constructs.
(A–M) C-terminal fusion constructs. For each fusion protein, the linker amino acid length is indicated after the name of the targeted organelle or fusion protein. (A) mTagBFP2-lamin B1-10; (B) mTagBFP2-CAAX-farnesyl-5; (C) mTagBFP2-endoplasmic reticulum-5 (calreticulin and KDEL); (D) mTagBFP2-fibrillarin-7; (E) mTagBFP2-light chain clathrin-15; (F) mTagBFP2-β-actin-7; (G) mTagBFP2-caveolin 1-10; (H) mTagBFP2-vinculin-22; (I) mTagBFP2-CAF1-10 (chromatin assembly factor); (J) mTagBFP2-Rab5a-7; (K) mTagBFP2-α-tubulin-18; (L) mTagBFP2-myosin-IIA-18; (M) mTagBFP2-PCNA-19. (N–X) N-terminal fusion constructs. (N) Cx26-mTagBFP2-7; (O) TfR-mTagBFP2-20 (transferrin receptor); (P) Golgi-mTagBFP2-7; (Q) zyxin-mTagBFP2-6;(R) VE cadherin-mTagBFP2-10; (S) mitochondria-mTagBFP2-7; (T) CENPB-mTagBFP2-22; (U) α-actinin-mTagBFP2-19; (V) c-src-mTagBFP2-7; (W) Lifeact-mTagBFP2-7; (X) vimentin-mTagBFP2-7. The cell line used for expressing mTagBFP2 fusion vectors was opossum kidney cortex proximal tubule epithelial cells (ATCC CRL-1840) in panel X, and human cervical adenocarcinoma cells (HeLa; ATCC CCL-2) in the remaining panels. The scale bar in each panel equals 10 µm.
Figure 5
Figure 5. Behavior of mTagBFP and mTagBFP2 as FRET donors.
(A) mTagBFP-mEGFP; (B) mTagBFP-mEmerald; (C) mTagBFP2-mEGFP; (D) mTagBFP2-mEmerald. Left column: emission spectra of FRET pairs with excitation at 405 nm before (black lines) and after (red lines) acceptor photobleaching. Columns 2 and 3: fixed cells in blue (Ex. 405 nm, Em. 445–470 nm) and green (Ex. 488 nm, Em. 500–535 nm) fluorescence channels after acceptor photobleaching in the square region (black square in the green channel). Column 4: FRET efficiency in fixed cells. Columns 5–6: live cells in blue/green channel before (column 5) and after (column 6) acceptor photobleaching are shown. Column 7: FRET efficiency in live cells. Colored scale bars (columns 4 and 7) show relative FRET efficiencies. See also Table 2.

Similar articles

See all similar articles

Cited by 82 articles

See all "Cited by" articles


    1. Piatkevich KD, Verkhusha VV. Advances in engineering of fluorescent proteins and photoactivatable proteins with red emission. Curr Opin Chem Biol. 2010;14:23–29. - PMC - PubMed
    1. Wu B, Piatkevich KD, Lionnet T, Singer RH, Verkhusha VV. Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization and dynamics. Curr Opin Cell Biol. 2011;23:310–317. - PMC - PubMed
    1. Heim R, Tsien RY. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence energy transfer. Curr Biol. 1996;6:178–182. - PubMed
    1. Heim R, Prasher DC, Tsien RY. Wavelength mutations and posttranslational autooxidation of green fluorescent protein. Proc Natl Acad Sci USA. 1994;91:12501–12504. - PMC - PubMed
    1. Tomosugi W, Matsuda T, Tani T, Nemoto T, Kotera I, et al. An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat Methods. 2009;6:351–353. - PubMed

Publication types


LinkOut - more resources