Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract.
Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.
Platis
Dimitris
D
Enzyme Technology Laboratory, Department of Agricultural Biotechnology, Agricultural University of Athens, Athens, Greece.
Labrou
Nikolaos E
NE
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D005591:Chemical Fractionation / Q000379:methods*; D044367:Phase Transition; D010710:Phosphates / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification*; D030821:Plants, Genetically Modified / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D014026:Nicotiana / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D010710:Phosphates; D010936:Plant Extracts; D011092:Polyethylene Glycols