ABSTRACT
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.
Subject(s)
Plant Proteins , Ribulose-Bisphosphate Carboxylase , Plant Proteins/genetics , Plant Proteins/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism , Proteomics/methods , Freezing , Photosynthesis/physiology , Plants/metabolism , Recombinant Proteins/metabolism , Plant Extracts , Plant Leaves/metabolism , Carbon Dioxide/metabolismABSTRACT
The effects of acute or chronic intake of boysenberry juice or artificial vinegar on blood pressure (BP) and endothelial function were investigated in spontaneous hypertensive rats (SHR). A single administration of boysenberry juice (BJ, equivalent to 0.5 mL/kg body weight) or artificial boysenberry juice vinegar (BJV, equivalent to 0.5 mL BJ and 0.10 g acetic acid/kg body weight) decreased both systolic blood pressure (SBP) and diastolic blood pressure (DBP) significantly. Reductions in SBP of the control group compared with the BJ and BJV groups reached maxima of -16.8±4.3 and -28.4±7.3 mmHg 8 h after administration, respectively. Chronic SBP- and DBP-lowering effects were also observed upon daily feedings of both BJ and BJV at 4 wk. No significant differences were found in SBP or DBP between respective acute and chronic intake of BJ and BJV, except for the decrease in DBP after 4 wk of BJV intake. This suggests that the polyphenol constituents in BJ and BJV likely play a major role in lowering SBP and DBP under these conditions and that acetic acid added to BJ exerts a DBP-lowering effect after 4 wk of BJV intake. The polyphenolic constituents of these beverages might elevate plasma NO concentration via aortic endothelial nitric oxide synthase activation, but the effects of chronic intake on blood pressure might also be at least partly mediated by the renin-angiotensin system. These results may help explain the beneficial effects of boysenberry intake on cardiovascular health, such as reduced blood pressure and improved endothelial function.
