Removal of plant endogenous proteins from tobacco leaf extract by freeze-thaw treatment for purification of recombinant proteins.

Plants are useful as a low-cost source for producing biopharmaceutical proteins. A significant hurdle in the production of recombinant proteins in plants, however, is the complicated process of removing plant-derived components. Removing endogenous plant proteins, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major photosynthetic plant enzyme that catalyzes photosynthesis through carboxylation and oxygenation, is important for the purification of recombinant plant proteins. In particular, RuBisCO accounts for 50% of the soluble leaf protein; thus, the removal of RuBisCO is critical for the purification of recombinant proteins from plant materials. An effective conventional method, known as freeze-thaw treatment, was developed for the removal of RuBisCO from Nicotiana benthamiana, which expresses recombinant green fluorescent protein (GFP). Crude extracts or supernatants were frozen at - 30 °C. Upon thawing, most of the RuBisCO was precipitated by centrifugation without significant inactivation and/or yield reduction of GFP. Based on the proteomics analysis, using this method, RuBisCO large and small subunits were reduced to approximately 10% and 20% of those of the unfrozen supernatant solutions, respectively, without the need for specific reagents or equipment. The proteomic analysis also revealed that many ribosomal proteins were removed from the extracts. This method improves the purification process of recombinant proteins from plant materials. Prolonged freezing damaged recombinant ß-glucuronidase (GUS), suggesting that the applicability of this treatment should be carefully considered for each recombinant protein.

Small-scale, high-throughput method for plant N-glycan preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

A simple, small-scale, and high-throughput method for preparation of plant N-glycans for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. This method entailed the extraction of soluble proteins, pepsin digestion, release of N-glycans by glycopeptidase A, and a three-step chromatographic purification process using cation exchange, anion exchange, and graphitized carbon. Homemade minicolumns using commercially available filter unit devices were used for N-glycan purification steps. All purification steps were designed to be easy. Using this method, N-glycans from 10-mg leaf samples of different plant species and only 2 µg of pure horseradish peroxidase were successfully purified.