Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

FP5

PRP AND NEURODEGENER Informe resumido

Project ID: QLG3-CT-2001-02353
Financiado con arreglo a: FP5-LIFE QUALITY
País: Italy

PrP 106-126 synthesis and purification

PrP106-126 was synthesised by stepwise solid-phase synthesis on an automated Applied Biosystems synthesiser model 433A at 0.1mM scale with hydroxymethylphenoxy (Wang-type HMP) resin from N-(9-fluorenyl) methoxycarbonyl (Fmoc) protected L-amino acid derivatives. Amino acids were activated by reaction with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluranium tetrafluoroborate. A capping step with acetic anhydride was included after the last coupling cycle of each amino acid.

The peptide was cleaved from the resin with a mixture of trifluoroacetic acid (TFA)/thioanisole/water/phenol/ethanedithiol 82.5:5:5:5:2.5 (v/v), precipitated with cold diethyl ether, and washed several times with the same solvent. PrP106-126 was purified by reverse-phase (RP)-HPLC on a semi-preparative C4 column (Symmetry 300, 19x150mm, particle size 7um, Waters, Japan) with a mobile phase of 0.1% TFA/water (eluent A) and 0.08% TFA/acetonitrile (ACN) (eluent B) using a linear gradient of 0-60% eluent B in 40min with a flow rate of 4ml/min. The fractions were collected, lyophilised and stored at -80°C. The identity of the peptide was verified by mass spectrometry (MS) using a Reflex IIITM MALDI mass spectrometer. A few µl of sample were mixed with an equal volume of a saturated solution of alpha-cyano-4-hydroxycinnamic acid in ACN/0.1% TFA 1:1 (v:v), and µl of the mixture was deposited on the MALDI target.

Chiesa, R., Fioriti, L., Tagliavini, F., Salmona, M., and Forloni, G. (2005). Cytotoxicity of PrP peptides. In Techniques in Prion Research, S. Lehmann, and J. Grassi, eds. (Basel, Birkhäuser Verlag).

Contacto

Roberto CHIESA, (Head of Department)
Tel.: +39-02-39014428
Fax: +39-02-3546277
Correo electrónico
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