Recovery and Characterization of Proteins from Plants: Aqueous Two-Phase System-Based Bioengineering Strategies
Aguilar Jiménez, Oscar A.
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The main drawback for general acceptance of plants as economically viable production systems is the lack of efficient initial concentration and separation procedures. In order to facilitate the general acceptance of plants as bioreactors, the establishment of efficient downstream operations is critical. It has been established that with the general knowledge of the molecular properties of contaminant proteins, the selection and design of suitable downstream strategies for recombinant proteins can be improved. The present dissertation addresses the potential use of quantitative 2D electrophoresis (2-DE) coupled with hydrophobic partitioning in aqueous two-phase systems (ATPS) for three-dimensional characterization of proteins from plant extracts. The application of this experimental approach to soybean proteins resulted in molecular characterization of proteins. Molecular weight (MW), isoelectric point (pI) and hydrophobicity were measured simultaneously and demonstrated that this technique can be a valuable tool for predictive design of recovery steps for recombinant proteins from plants. The extension of this experimental approach in alfalfa green tissue extracts containing a model recombinant protein provided additional information on the molecular properties of the main host proteins that will allow the design of pre-fractionation and purification methods to facilitate its recovery from alfalfa extracts. As a result of the application of this three-dimensional characterization technique to soybean and alfalfa protein extracts, more efficient downstream strategies could be designed for recovery of recombinant proteins, facilitating the future adoption of plants as a production system.