mBeRFP, an improved large stokes shift red fluorescent protein

Abstract

Herein, we describe the generation of a monomeric large Stokes shift (LSS) red fluorescent protein, mBeRFP, with excitation and emission peaks at 446 and 615 nm, respectively. Compared with two previously reported LSS-RFPs (mKeima and LSS-mKate2), mBeRFP is approximately three times brighter. In addition, mBeRFP is characterized by improved photostability, rapid maturation, an extended lifetime, and a monomeric nature. Additionally, mBeRFP can be paired with the Alexa 647 dye as a FRET donor to detect caspase 3 activity. This FRET pair has an extremely dynamic range and a large Förster radius (approximately 6.5 nm). To demonstrate the applicability of mBeRFP for imaging in living cells, we performed dual-color imaging of mBeRFP and CFP simultaneously excited by a single excitation source, and we demonstrated that these fluorescent proteins allow the clear visualization of the dynamics of Bax during cancer cell apoptosis. Thus, mBeRFP appears to be particularly useful for cellular imaging applications.

Figure 1. Characterization of the purified variants.

(A) The normalized excitation (solid line) and emission (dashed line) spectra of mBeRFP. (B) Normalized curve of the maturation times of mBeRFP (solid line), mLSS-mKate2 (dashed line), and mKeima (dotted line). (C) Absorption spectra at various pH values (4–10). (D) Photobleaching times of mBeRFP (solid line), mLSS-mKate2 (dashed line), and mKeima (dotted line).

Figure 2. Gel filtration spectra of mKate (A), mKate V97S (B), and mBeRFP (C).

Figure 3. mBeRFP was paired with Alexa 647 to form a FRET donor/acceptor pair.

(A) The fluorescence spectra of mBeRFP (solid line) and Alexa 647 (dashed line). (B) The mBeRFP-DEVD-SNAP 647 protein was digested by caspase 3 in vitro at 37°C. The spectral sign was recorded at the 0, 40, and 90 min time points.

Figure 4. Use of mBeRFP in the fluorescent imaging of living cells.

(A) Left panel: pcDNA3.0-mBeRFP expressed in a HeLa cell. Right panel: The chimeric protein mBeRFP-α-tubulin (human) expressed in a B16 cell (mouse melanoma cell line). (B) Upper panel: excitation spectra of CFP (cyan line) and mBeRFP (red line). Lower panel: emission spectra of CFP (cyan line) and mBeRFP (red line). (C) The chimeric proteins mCerulean-Bax and mt-mBeRFP were co-expressed in HeLa cells. The cells were exposed to 30 µg/mL cisplatin to induce apoptosis. Dual fluorescent signals were recorded after the cells were treated (scale bar = 10 µm).