J. Sep. Sci. 2009, 32, 3223 – 3231 O. Aguilar et al. 3223 Oscar Aguilar 1 Charles E. Glatz 2 Marco Rito-Palomares 1 1 Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey, Campus Monterrey, Monterrey, NL, MØxico 2 Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, USA Original Paper Characterization of green-tissue protein extract from alfalfa (Medicago sativa) exploiting a 3-D technique There is a growing interest of pharmaceutical companies for plant-based production systems. To facilitate the general acceptance of plants as bioreactors, the establish- ment of efficient downstream operations is critical. It has been proposed that a bet- ter understanding of the properties of the contaminant proteins can benefit down- stream processing design and operation. The coupled application of 2-DE with aque- ous two-phase partitioning has been suggested as a practical 3-D method to charac- terize potential contaminant proteins from plant extracts. The application of this novel 3-D approach to a complex protein extract from alfalfa (Medicago sativa) contain- ing a model recombinant protein (human granulocyte colony stimulating factor (hG-CSF)) resulted in the quantification of 55 protein spots. The 3-D properties (M r ,pI, and K p ) obtained for 17 proteins comprising 69% of the alfalfa proteins, allowed the proposal of a prefractionation step as well as the identification of the target mole- cule (rG-CSF) from bulk of alfalfa proteins. The information obtained from this exper- imental approach was useful for the identification of the potential contaminant pro- teins that will occur in alfalfa when this plant is used as a host for recombinant pro- teins. Additionally, this method will assist in the design of adequate purification strategies for recombinant proteins expressed in alfalfa green tissue. Keywords: Alfalfa protein / Aqueous two-phase systems / 2D-electrophoresis / G-CSF / Proteomics / Received: March 24, 2009; revised: May 14, 2009; accepted: May 15, 2009 DOI 10.1002/jssc.200900184 1 Introduction A wide number of pharmaceutical proteins have been produced in a variety of plant species (including tobacco, potato, rice, soybean alfalfa, tomato, and lettuce) reflect- ing the interest of biotechnology companies to benefit from the advantages of plant-based production systems [1, 2]. During the design of a recombinant protein pro- duction process, selection of the most adequate expres- sion system as well as an efficient extraction and purifi- cation strategy to maximize recovery of target protein, represent the major aspects to be considered. Down- stream processing costs typically contribute 80% of the total. Therefore, efficient and robust processing strat- egies are essential [3]. In this context, the use of aqueous two-phase systems (ATPSs)-based strategies have resulted in the establishment of protocols for the recovery and purification of biological compounds [4 – 7]. ATPSs have also been used for the understanding of chemical proper- ties and behavior of proteins in solution [8]. It is clear that a better understanding of the properties of the contaminant proteins can benefit downstream processing design and operation [9, 10]. Proteomic tools like MS and 2-DE have become common techniques to accurately detect and examine protein composition from a variety of plant hosts. These techniques provide useful information on the molecular properties of com- plex mixtures that can be exploited for the optimization and better design of downstream strategies [11]. A 3-D technique for the molecular characterization of corn germ protein extracts was recently reported by Gu and Glatz [9]. It was based on the coupled application of aqueous two-phase partitioning to measure hydropho- bicity in terms of the partition coefficient of the proteins (K p ), and 2-DE to evaluate molecular weight (M r ) and pI of individual proteins [9]. The 3-D information obtained for each protein (M r ,pI, and hydrophobicity) was used as a Correspondence: Dr. Marco Rito-Palomares, Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnolo- gía, Tecnológico de Monterrey, Campus Monterrey, Ave. Euge- nio Garza Sada 2501 Sur, Monterrey, NL 64849, MØxico E-mail: mrito@itesm.mx Fax: +52-81-8328-4136 Abbreviations: ATPS, aqueous two-phase system; LAC, a-lactalbu- min; LOD, limit of detection; LYS, lysozyme; rG-CSF, recombinant granulocyte colony stimulating factor; RNA, ribonuclease A i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com