IDENTIFICATION OF A RECOMBINANT SYNAPTOBREVIN-THIOREDOXIN FUSION PROTEIN BY CAPILLARY ZONE ELECTROPHORESIS USING LASER-INDUCED FLUORESCENCEDETECTION

Capillary zone electrophoresis (CZE) was utilized to identify a synapt obrevin-thioredoxin fusion protein (TSB-51). TSB-51 is a substrate for cleavage by botulinum toxin B at the Q(76)-F(77) site, TSB-51 was der ivatized with a fluorophore, CBQCA 3-(4-carboxy-benzoyl)-2-quinoline-c arboxaldehyde], for 4 h at room temperature, Optimal conditions for CZ E separation of the TSB-51-CBQCA complex were determined: buffer (sodi um berate), pH (9.0), applied voltage (25 kV), temperature (25 degrees C) and forward polarity. SDS-PAGE showed that TSB-51 had a molecular mass of similar to 19 kDa. The protein was transferred to PVDF membran e and sequenced by the Edman degradation method verifying the first tw elve amino acids as SDKIIHLTDDSF, TSB-51 was also collected during CZE separation and subsequently sequenced yielding the first three amino acids as SDK. This CZE-LIF method coupled with the CBQCA derivatizatio n, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilizatio n of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botu linum toxin B on TSB-51.