Performance and use

TurboGFP can be expressed and detected in a wide range of organisms including cold-blooded animals. Mammalian cells transiently transfected with TurboGFP expression vectors produce bright fluorescence in 8-10 hrs after transfection. No cytotoxic effects or visible protein aggregation are observed. TurboGFP can be used in multicolor labeling applications with blue, true-yellow, red, and far-red fluorescent dyes.

	TurboGFP expression in transiently transfected mammalian cells.

TurboGFP expression in transiently transfected mammalian cells.

TurboGFP suitability to generate stably transfected cells has been proven by Marinpharm company.

Despite its dimeric structure, TurboGFP performs well in some fusions. However, for protein labeling applications we recommend using specially optimized monomeric TagFPs.


TurboGFP expression in stably transfected mammalian cell lines. (A) Human cervix carcinoma HeLa cells with TurboGFP in cytoplasm; (B) C2C12 mouse myoblasts with TurboGFP in cytoplasm; (C) PC-12 rat phaeochromocytoma cells with TurboGFP in cytoplasm; (D) PC-12 rat phaeochromocytoma cells with TurboGFP in cytoplasm after the addition of nerve growth factor (cells differentiate irreversibly into neuron-like cells); (G) Walker 256 rat tumour cells with TurboGFP in cytoplasm; (H) M3-mouse melanoma with TurboGFP in cytoplasm; (I) 3T3 mouse fibroblast with TurboGFP in cytoplasm; (J) T24 human bladder carcinoma with TurboGFP in cytoplasm; (E) Chinese Hamster Ovary Cells CHO-K1 with TurboGFP in cytoplasm; (F) HeLa cells expressing TurboGFP-fibrillarin fusion; (K) HeLa cells expressing mitochondria-targeted TurboGFP; (L) T24 human bladder carcinoma expressing mitochondria-targeted TurboGFP. Images were kindly provided by Dr. Christian Petzelt (Marinpharm).

TurboGFP expression in stably transfected mammalian cell lines.

(A) Human cervix carcinoma HeLa cells with TurboGFP in cytoplasm; (B) C2C12 mouse myoblasts with TurboGFP in cytoplasm; (C) PC-12 rat phaeochromocytoma cells with TurboGFP in cytoplasm; (D) PC-12 rat phaeochromocytoma cells with TurboGFP in cytoplasm after the addition of nerve growth factor (cells differentiate irreversibly into neuron-like cells); (G) Walker 256 rat tumour cells with TurboGFP in cytoplasm; (H) M3-mouse melanoma with TurboGFP in cytoplasm; (I) 3T3 mouse fibroblast with TurboGFP in cytoplasm; (J) T24 human bladder carcinoma with TurboGFP in cytoplasm; (E) Chinese Hamster Ovary Cells CHO-K1 with TurboGFP in cytoplasm; (F) HeLa cells expressing TurboGFP-fibrillarin fusion; (K) HeLa cells expressing mitochondria-targeted TurboGFP; (L) T24 human bladder carcinoma expressing mitochondria-targeted TurboGFP.

Images were kindly provided by Dr. Christian Petzelt (Marinpharm).

TurboGFP maturation kinetics: TurboGFP allows monitoring the activity from early promoters. It matures noticeably faster than EGFP and most other fluorescent proteins. This difference in performance is illustrated here using both in vitro analysis of TurboGFP and EGFP refolding and maturation kinetics and in vivo examination of the developing Xenopus embrios expressing either TurboGFP or EGFP.

Refolding and maturation kinetics of TurboGFP and some other fluorescent proteins *in vitro*
Fluorescent protein	Refolding half-time, s	Maturation half-time,s	kox (10^-4s^-1)	Reference
Samples of fluorescent proteins were heated to 95°C in denaturation solution (8 M urea, 1 mM DTT) for 4 min. Refolding reactions were initiated upon 100-fold dilution into the renaturation buffer (35 mM KCl, 2 mM MgCl₂, 50 mM Tris pH 7.5, 1 mM DTT). In maturation assay, 5 mM freshly dissolved dithionite was added to the denaturation solution [Reid and Flynn, 1997]. Due to the instability of dithionite at high temperatures, to provide for complete chromophore reduction the sample was cooled to 25°C and the addition of 5 mM dithionite followed by heating to 5°C were repeated. Protein refolding and maturation were followed by measuring the recovery of fluorescence using Varian Cary Eclipse Fluorescence Spectrophotometer, chamber temperature maintained at 25°C. Maturation rate constants (kox) were determined by computer-fitting the kinetic data to the first order exponential decay (Origin 6.0).
EGFP	90.6	3915	1.77	Evdokimov et al., 2006
Venus	46.2	4076	1.70	Kremers et al., 2006
SYFP2	69.3	3300	2.10	Kremers et al., 2006
TurboGFP	11.0	1468	4.72	Evdokimov et al., 2006

		Comparison of EGFP (violet lines) and TurboGFP (green lines) refolding and maturation speed in vitro. Normalized fluorescence recovery plots are shown. (A) — refolding kinetics; (B) — chromophore maturation kinetics.

Comparison of EGFP (violet lines) and TurboGFP (green lines) refolding and maturation speed in vitro.

Normalized fluorescence recovery plots are shown.

(A) — refolding kinetics;

(B) — chromophore maturation kinetics.

		In vivo comparison of TurboGFP and EGFP maturation in developing Xenopus embryos Vectors encoding TurboGFP and EGFP under the control of CMV promoter were microinjected into animal poles of Xenopus embryos at the stage of two blastomeres. Living embryos were then photographed from the animal pole at the middle and late gastrula stages. Experimental data were presented by Dr. A. Zaraisky, Institute of Bioorganic Chemistry, RAS (Moscow, Russia).

In vivo comparison of TurboGFP and EGFP maturation in developing Xenopus embryos

Vectors encoding TurboGFP and EGFP under the control of CMV promoter were microinjected into animal poles of Xenopus embryos at the stage of two blastomeres. Living embryos were then photographed from the animal pole at the middle and late gastrula stages.

Experimental data were presented by Dr. A. Zaraisky, Institute of Bioorganic Chemistry, RAS (Moscow, Russia).