Application of free-flow electrophoresis to the purification of trichosanthin from a crude product of acetone fractional precipitation

The application of free-flow electrophoresis (FFE) to the purification of trichosanthin (TCS) from a crude product of acetone fractional precipitation was investigated. An electrophoresis technique, combining field step electrophoresis (FSE) and zone electrophoresis (ZE) to a one-step procedure, was optimized until a satisfactory purification factor (1.35), high resolution, and purity (>99%) were achieved. Testing several separation buffer systems revealed that a throughput of 14.2 mg/h can be obtained when the very basic TCS (pI 10.1) was dissolved and electrophoresed in a phosphate buffer system of pH 4. The purity of electrophoresed trichosanthin was proved by a variety of analytical methods, such as sodium dodecyl sulfate (SDS)-gel electrophoresis, capillary isoelectric focusing (CIEF), and sequencing of N- and C-termini. The high purity and large throughput achieved at low cost by using FFE indicates that this method can be employed for TCS purification.