Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey, Campus Monterrey, Monterrey, NL, México.
J Sep Sci. 2009 Sep;32(18):3223-31. doi: 10.1002/jssc.200900184.
There is a growing interest of pharmaceutical companies for plant-based production systems. To facilitate the general acceptance of plants as bioreactors, the establishment of efficient downstream operations is critical. It has been proposed that a better understanding of the properties of the contaminant proteins can benefit downstream processing design and operation. The coupled application of 2-DE with aqueous two-phase partitioning has been suggested as a practical 3-D method to characterize potential contaminant proteins from plant extracts. The application of this novel 3-D approach to a complex protein extract from alfalfa (Medicago sativa) containing 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 molecule (rG-CSF) from bulk of alfalfa proteins. The information obtained from this experimental approach was useful for the identification of the potential contaminant proteins that will occur in alfalfa when this plant is used as a host for recombinant proteins. Additionally, this method will assist in the design of adequate purification strategies for recombinant proteins expressed in alfalfa green tissue.
制药公司对基于植物的生产系统越来越感兴趣。为了促进植物作为生物反应器的普遍接受，建立高效的下游操作至关重要。有人提出，更好地了解污染物蛋白的特性可以有利于下游加工设计和操作。已经有人建议将 2-DE 与双水相分配相结合，作为一种实用的 3D 方法来表征植物提取物中的潜在污染物蛋白。该新型 3D 方法应用于含有模型重组蛋白（人粒细胞集落刺激因子（hG-CSF））的复杂苜蓿（Medicago sativa）蛋白提取物中，结果定量了 55 个蛋白点。对于由 69%苜蓿蛋白组成的 17 种蛋白，获得的 3D 特性（M(r)、pI 和 K(p)）允许提出预分级步骤，以及从苜蓿蛋白混合物中鉴定出目标分子（rG-CSF）。从这种实验方法获得的信息有助于鉴定当苜蓿被用作重组蛋白的宿主时，在苜蓿中可能出现的潜在污染物蛋白。此外，该方法将有助于设计在苜蓿绿色组织中表达的重组蛋白的适当纯化策略。
