Identification of a recombinant synaptobrevin-thioredoxin fusion protein by capillary zone electrophoresis using laser-induced fluorescence detection.
Capillary zone electrophoresis (CZE) was utilized to identify a synaptobrevin-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 derivatized with a fluorophore, CBQCA [3-(4-carboxy-benzoyl)-2-quinoline-carboxaldehyde], for 4 h at room temperature. Optimal conditions for CZE separation of the TSB-51-CBQCA complex were determined: buffer (sodium borate), 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 approximately 19 kDa. The protein was transferred to PVDF membrane and sequenced by the Edman degradation method verifying the first twelve 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 derivatization, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilization of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botulinum toxin B on TSB-51.
Asermely
K E
KE
Pharmacology Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, USA.
Broomfield
C A
CA
Nowakowski
J
J
Courtney
B C
BC
Adler
M
M
D000595:Amino Acid Sequence; D001565:Benzoates; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D011804:Quinolines; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013879:Thioredoxins / Q000032:analysis*
D001565:Benzoates; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011804:Quinolines; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C069617:3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde; D013879:Thioredoxins