Methods for the determination of the biological activities of neurotoxin polypeptides

Abstract

The present invention pertains to a method for determining the biological activity of a neurotoxin, the method comprising the steps of: (a) expressing a fusion protein comprising (i) an anchor protein, (ii) a reporter protein and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, in neurotoxin-sensitive cells; (b) incubating the neurotoxin-sensitive cells of (a) with a neurotoxin and cultivating the cells under conditions which allow the neurotoxin to exert its biological activity; (c) permeabilizing the neurotoxin-sensitive cells of (b) under conditions which allow the release of the reporter protein but not the release of the anchor protein from the permeabilized neurotoxin-sensitive cells; and (d) quantifying the activity of the reporter protein released from the cells, thereby determining the biological activity of the neurotoxin. In addition, the invention relates to a fusion protein comprising (i) an anchor protein, (ii) a reporter protein, and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, for determining the biological activity of a neurotoxin, in neurotoxin-sensitive cells. Further encompassed by the present invention is a kit comprising the fusion protein of the invention. Finally, the invention pertains to the use of a fusion protein of the invention for determining the biological activity of a neurotoxin, in neurotoxin-sensitive cells.

Claims

1. A method for determining the biological activity of a neurotoxin, the method comprising the steps of: (a) expressing a fusion protein comprising (i) an anchor protein, (ii) a reporter protein and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, in neurotoxin-sensitive cells; (b) incubating the neurotoxin-sensitive cells of (a) with a neurotoxin and cultivating the neurotoxin-sensitive cells under conditions which allow the neurotoxin to exert its biological activity; (c) permeabilizing the neurotoxin-sensitive cells of (b) under conditions which allow the release of the reporter protein but not the release of the anchor protein from the permeabilized neurotoxin-sensitive cells; and (d) quantifying the activity of the reporter protein released from the permeabilized neurotoxin-sensitive cells of (c), thereby determining the biological activity of the neurotoxin.

2. The method of claim 1, wherein the neurotoxin-sensitive cells are derived from tumor cell lines, primary cells, stem cells or induced pluripotent stem cells.

3. The method of claim 1, wherein the anchor protein is a membrane protein selected from the group consisting of a choline transporter, H1-receptor, G protein-coupled receptor (GPCR) and SV2.

4. The method of claim 1, wherein the neurotoxin cleavage site is selected from the group consisting of a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT cleavage site.

5. The method of claim 1, wherein the reporter protein is an enzyme selected from the group consisting of luciferase, alkaline phosphatase, beta-galactosidase, and horseradish peroxidase (HRP) or a fluorescent protein selected from the group consisting of GFP, YFP, BFP and RFP.

6. The method of claim 1, wherein a hemolysin is used for the permeabilization of the neurotoxin-sensitive cells.

7. The method of claim 6, wherein the hemolysin is selected from the group consisting of streptolysin O, perfringolysin O, pneumolysin, a bacterial hemolysin and a pore-forming toxin from snakes or spiders.

8. The method of claim 1, wherein the fusion protein comprises a fusion protein selected from choline transporter-GFP-SNAP-25-luciferase, H1-receptor-SNAP-25-luciferase and H1-receptor-SNAP-25-HRP.

9. The method of claim 1, wherein the quantification of the activity of the reporter protein comprises the standardization of the activity of the reporter protein.

10. The method of claim 9, wherein the standardization of the activity of the reporter protein is carried out by determining the residual reporter protein activity of the non-cleaved fusion protein remaining in or at the neurotoxin-sensitive cells or the total reporter protein activity of the fusion protein.

11. A fusion protein consisting of (i) an anchor protein, (ii) a reporter protein, and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, for determining the biological activity of a neurotoxin, in neurotoxin-sensitive cells.

12. The fusion protein of claim 11, wherein the anchor protein is selected from the group consisting of choline transporter, H1-receptor, G protein-coupled receptor (GPCR) and SV2; the reporter protein is an enzyme selected from the group consisting of luciferase, alkaline phosphatase, beta-galactosidase and horseradish peroxidase or a fluorescent protein selected from the group consisting of GFP, YFP, BFP and REP; and the neurotoxin cleavage site is selected from the group consisting of a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT cleavage site.

13. The fusion protein of claim 11, wherein the fusion protein comprises a fusion protein selected from choline transporter-GFP-SNAP-25-luciferase, H1-receptor-SNAP-25-luciferase and H1-receptor-SNAP-25-HRP.

14. A kit comprising the fusion protein of claim 11.

Description

EXAMPLE

(1) The DNA molecule with the sequence shown in SEQ ID NO: 5 encoding the amino acid sequence shown in SEQ ID NO: 2 (choline transporter-GFP-SNAP-25-luciferase; CHT-GFP-SNAP-LUC) is synthesized de novo, then cloned into pcDNA 3.1 vector (Life Technologies). Further the plasmid (DNA vector with inserted construct) is amplified, extracted and purified. DNA synthesis, cloning, amplification and isolation are performed by GeneArt, Life technologies.

(2) The plasmid is transfected into SH-SY5Y cells (ATCC CRL-2266) using Lipofectamine 2000 (Life Technologies). Cell culture and transfection are performed according to the provider's instructions.

(3) After transfection of the plasmid, the cells are cultivated in the presence of Gentamicine in order to selectively grow plasmid containing cells. As soon as the culture reaches approx. 5 million cells, a single cell cloning is performed using the method of limited dilution. By this, approx. 400 clones are created and tested for adhesion quality, growth rates, plasmid expression rate and sensitivity for BoNT/A. The expression rate is determined by measuring the GFP fluorescence while the BoNT/A sensitivity is tested by the western blot SNAP-25 cleavage assay, according to Pellett S, Tepp W H, Clancy C M, Borodic G E, Johnson E A. FEBS Lett. 2007 Oct. 16; 581(25): 4803-8. From the clones showing good adhesion and growth rates and BoNT/A sensitivities comparable or better than SH-SY5Y parental cells, those with the highest GFP expression rates are selected and stored in cell banks.

(4) For the BoNT/A reporter assay, the cells are seeded on 96-well plates followed by differentiation according to Rasetti-Escargueil et al. (Enhanced sensitivity to Botulinum type A neurotoxin of human neuroblastoma SH-SY5Y cells after differentiation into mature neuronal cells by C. Rasetti-Escargueil, C. B. Machado, R. Preneta-Blanc, R. A. Fleck, D. Sesardic The Botulinum J. (TBJ), Vol. 2, No. 1, 2011). The differentiated cells are incubated for 72 h with BoNT/A diluted in culture medium to final concentrations between 0.01 and 10 pM in logarithmic steps. After incubation, the cells are washed once with PBS (10 mM Sodium Phosphate, 0.9% NaCl, pH 7.4) and permeabilized with 501 Streptolysin 0 solution (10 mM Sodium Phosphate, 0.9% NaCl, 10 mM Dithiothreitol, 50 U/mL Streptolysin O, pH 7.4). After 15 min incubation at 37 C., the supernatant of each well is transferred into a white 96-well plate and the luciferase activity is determined, according to the provider's instructions (Sigma Aldrich LUC1-1KT). The cells remaining on the culture plates are washed once with PBS and the GFP fluorescence is measured in a plate reader. For each single well the Luc/GFP ratio is calculated by dividing the measured signal values. The BoNT/A biological activity is calculated by comparing the dose-response-curve of the unknown sample with the respective curve of a reference standard e.g. by parallel line assay.