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.
Song
J F
JF
Shanghai Institute of Biochemistry, Chinese Academy of Sciences.
Liu
T
T
Shen
X
X
Wu
G D
GD
Xia
Q C
QC
D000096:Acetone / Q000737:chemistry*; D000595:Amino Acid Sequence; D002021:Buffers; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods*; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D004983:Ethanolamines; D005590:Fractional Precipitation; D010446:Peptide Fragments / Q000302:isolation & purification; D010710:Phosphates; D010940:Plant Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D015978:Trichosanthin / Q000302:isolation & purification*
D002021:Buffers; D004983:Ethanolamines; D010446:Peptide Fragments; D010710:Phosphates; D010940:Plant Proteins; D000096:Acetone; D012967:Sodium Dodecyl Sulfate; D015978:Trichosanthin; C009546:triethanolamine