Purification and stabilization of ricin B from tobacco hairy root culture medium by aqueous two-phase extraction.
Ricin B (RTB), the non-toxic lectin subunit of ricin, is a promising mucosal adjuvant and carrier for use in humans. RTB fusion proteins have been expressed in tobacco hairy root cultures, but the secreted RTB component of these proteins was vulnerable to protease degradation in the medium. Moreover, castor bean purified RTB spiked into tobacco hairy root culture media showed significant degradation after 24 h and complete loss of product after 72 h. Aqueous two-phase extraction (ATPE) was tested for fast recovery of RTB not only to partially purify the protein but also to improve its stability. Two different polyethylene glycol (PEG)/salt/water systems including PEG/potassium phosphate and PEG/sodium sulfate, were studied. RTB was shown to be favorably recovered in PEG/sodium sulfate systems. Statistical analysis indicated that the ionic strength of the system and the sodium sulfate concentration were important in optimizing the partition coefficient of RTB. A selectivity of almost three could be achieved for RTB in optimized systems, and RTB partitioned in the PEG-rich phase exhibited extended stability. Therefore, ATPE was shown to be effective in initial recovery/purification and stabilization of RTB and may hold promise for other unstable secreted proteins from hairy root culture.
Zhang
Chenming
C
Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210 Seitz Hall, Blacksburg, VA 24061, USA. cmzhang@vt.edu
Medina-Bolivar
Fabricio
F
Buswell
Scott
S
Cramer
Carole L
CL
D003470:Culture Media; D004355:Drug Stability; D018517:Plant Roots / Q000235:genetics*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000302:isolation & purification; D012276:Ricin / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility; D014026:Nicotiana / Q000235:genetics*
D003470:Culture Media; D011994:Recombinant Proteins; D012276:Ricin