Recombinant aprotinin produced transgenic corn seed: Extraction and purification studies

Expression in transgenic plants is potentially one of the most economical systems for large-scale production of valuable peptide and protein products. However, the downstream processing of recombinant proteins produced in plants has not been extensively studied. In this work, we studied the extraction and purification of recombinant aprotinin, a protease inhibitor used as a therapeutic compound, produced in transgenic corn seed. Conditions for extraction from transgenic corn meal that maximize aprotinin concentration and its fraction of the total soluble protein in the extract were found: pH 3.0 and 200 mM NaCl. Aprotinin, together with a native corn trypsin inhibitor (CTI), was captured using a tryspin-agarose column. These two inhibitors were separated using an agarose-IDA-Cu2+ column that proved to efficiently absorb the CTI while the recombinant aprotinin was collected in the flowthrough with purity of at least 79%. The high purity of the recombinant aprotinin was verified by SDS-PAGE and N-terminal sequencing. The overall recombinant aprotinin recovery yield and purification factor were 49% and 280, respectively. Because CTI was also purified, the recovery and purification process studied has the advantage of possible CTI co-production. Finally, the work presented here introduces additional information on the recovery and purification of recombinant proteins produced in plants and corroborates with past research on the potential use of plants as biorreactors. (C) 2002 Wiley Periodicals, Inc.80326827

Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere

Transgenic plants containing a chimeric gene construct that facilitates the exudation of proteins from roots offer novel approaches for modification of the rhizosphere and production of relatively pure recombinant proteins. The aim of this study was to develop alfalfa (Medicago sativa L.) plants that exude a heterologous recombinant protein into the rhizosphere. Alfalfa transformed with a fungal endochitinase (ech42) cDNA fused in frame to the signal peptide of a white lupin acid phosphatase and under the control of the cassava vein mosaic virus (CsVMV) promoter expressed increased chitinase activity in vegetative organs and root exudates. Chitinase activity in root exudates of transgenic alfalfa was 7.5–25.7 times higher than in the untransformed Regen-SY plants. Chitinase enzyme activity was accompanied by increased synthesis of mRNA and protein in transformed plants. By comparison, untransformed and vector only transformed plants displayed no expression of recombinant protein and mRNA. A single band of the expected molecular weight was present only in western blots of root exudates of transgenic alfalfa plants. The secreted endochitinase enzyme not only retained its lytic activity against glycol chitin but also showed antifungal activity by inhibition of spore germination of two fungal pathogens. Exudation of recombinant proteins from roots may offer alternative uses for alfalfa in the production of value-added biopharmaceuticals and may influence microbes or modify soil nutrient availability near plant roots.Mesfin Tesfaye, Matthew D. Denton, Deborah A. Samac & Carroll P. Vanc

Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences

Over the past decade, several high-value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds(1,2). Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris)(3) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single-chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops(5,6). In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large-scale production