Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 83:286–293 (2008) Processing of soybean (Glycine max) extracts in aqueous two-phase systems as a first step for the potential recovery of recombinant proteins Oscar Aguilar and Marco Rito-Palomares * Centro de Biotecnolog´ ıa, Departamento de Biotecnolog´ ıa e Ingenier´ ıa de Alimentos, Tecnol ´ ogico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, M ´ exico Abstract BACKGROUND: The potential use of plants as production systems to establish bioprocesses has been established over the past decade. However, the lack of efficient initial concentration and separation procedures affect the generic acceptance of plants as economically viable systems. In this context the use of aqueous two-phase systems (ATPS) can provide strategies to facilitate the adoption of plants as a base for bioprocesses. Among the crops, soybeans (Glycine max) represent an attractive alternative since potentially they can produce high levels of recombinant protein. In this paper the processing of fractionated soybean extracts using ATPS is evaluated as a first step to recover recombinant proteins expressed in plants, using β -glucuronidase (GUS; E.C. 3.2.1.31) as a model protein. RESULTS: The evaluation of the effect of system parameters provided the conditions under which the contaminant proteins from fractionated soybean extracts and GUS concentrated in opposite phases. A PEG 600/phosphate system comprising 14.5% (w/w) polyethylene-glycol (PEG), 17.5% (w/w) phosphate, a volume ratio (Vr) equal to 1.0, and a system pH of 7.0 resulted in the potential 83% recovery of GUS from the complex mixture and an increase in purity of 4.5-fold after ATPS. CONCLUSIONS: The findings reported here demonstrate the potential of ATPS to process fractionated soybean extract as a first step to isolate and purify a recombinant protein expressed in soybeans. The proposed approach can simplify the way in which recombinant proteins expressed in plants can be recovered.  2007 Society of Chemical Industry Keywords: aqueous two-phase systems; protein recovery; soybean extracts INTRODUCTION There is considerable interest in the development of biotechnological processes that exploit the use of plants as a host for producing recombinant proteins. The potential use of plants as bioreactors has been established over the past decade. Transgenic plants are a potentially inexpensive system for the large-scale production of recombinant proteins for use in the pharmaceutical, agricultural and industrial sectors. 1–3 The advantages of using transgenic plants include low cost and flexibility in large-scale production, the presence of natural storage organs such as seeds and tubers, and existing technology to harvesting and processing of plant material. 2 However, the lack of efficient initial concentration and separation procedures affect the potential generic acceptance of plants as economically viable systems. One of the main drawbacks of the seed-based technology is the inherent need for the plant to generate the flower in order to produce the seed, raising bio-safety concerns regarding avoiding the release of pollen to the environment and increasing the cost of production due to the need for containment and longer harvesting times. 4 The selection of an adequate crop for the production of recombinant proteins is affected by numerous aspects that contribute to the success and final cost of the products. The general alternatives include maize (Zea mays), rice (Oryza sativa), canola (Brassica sp.), tobacco (Nicotiana tabacum), peas (Pisum sativum) and soybean (Glycine max). Canola, maize and soy have been regarded as the most likely production systems for commercial application of plants as bioreactors. 5 Maize is preferred over other plant hosts due to its lower hydro-soluble protein content. Extensive research on the potential recovery of recombinant proteins expressed in this particular crop has been documented. 6,7 In the particular case of canola, compared to other crops, a greater fraction * Correspondence to: Marco Rito-Palomares, Centro de Biotecnolog´ ıa, Departamento de Biotecnolog´ ıa e Ingenier´ ıa de Alimentos, Tecnol ´ ogico de Monterrey E-mail: mrito@itesm.mx (Received 9 March 2007; revised version received 10 August 2007; accepted 18 August 2007) Published online 11 December 2007; DOI: 10.1002/jctb.1805  2007 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2007/$30.00