Supercharged green fluorescent proteins as bimodal reporter genes for CEST MRI and optical imaging

Abstract

Superpositively charged mutants of green fluorescent protein (GFP) demonstrated a dramatically improved chemical exchange saturation transfer (CEST) MRI contrast compared to their wild type counterparts. The mutants +36 GFP and +48 GFP were successfully expressed in mammalian cells and retained part of their fluorescence, making them a new potential bimodal reporter gene.

Figure 1

CEST MRI of GFP proteins. (a) Fluorescence of the examined GFP proteins and their purity, as determined by SDS-polyacrylamide gel electrophoresis. (b) MTR_asym maps obtained for a saturation pulse at Δω = 1.8 ppm frequency offset. Shown is the dependency of CEST contrast on B₁ power. CEST data from 1.25 mg mL⁻¹ pure protein solutions were acquired at 11.7 T, 37 °C, pH = 7.2, and B₁ = 4000 ms.

Figure 2

MTR_asym plots of GFP proteins as a function of applied saturation pulse (B₁) power: (a) 2.4 µT; (b) 3.6 µT; (c) 4.7 µT; and (d) 7.2 µT. Data from 1.25 mg mL⁻¹ pure protein solutions were acquired at 11.7 T, 37 °C, pH = 7.2, and B₁ = 4000 ms. N = 7 for each sample, error bars represent standard deviation.

Figure 3

Fluorescent microscopy images of human embryonic kidney cells (HEK293T) without transfection (a) and 24 h after transfection with optimized genes encoding for (b) wt, (c) +36, and (d) +48. Bar: 100 µm.