| Transgenic rice is an ideal bioreactor for large-scale production of recombinant proteins including pharmaceuticals and industrial enzymes. Recombinant proteins expressed transgenic rice seeds are produced with many benefits, including high accumulation of recombinant protein, lower production cost, high level of protein stability, stored for long periods of time, easy control of production scale, and low risk of contamination by human and animal pathogens. As the transgenic plant platform continues to mature, research and development interests will likely shift from upstream to downstream processing to reduce the total operating cost. Thus, the development of efficient and low-cost isolation and purification process of recombinant proteins with commercial potential is the future direction and research needs.Cellulosic ethanol, which is abundant and renewable, provides a promising alternative for biofuel requirements. One major technical hurdle for economically viable cellulosic ethanol is the need to reduce the costs of enzymes for the conversion of biomass to fermentable sugars. Genetic engineering technology offers great potential to decrease the costs of cellulases production. The transgenic rice seed is an attractive expression platform and holds significant advantages such as high protein yields and stable storage of target proteins. The thermostable endo-1,4-β-glucanase (E1) from Acidothermus cellulolyticus, is a useful enzyme for commercial hydrolysis of cellulose into glucose. Thus the goal of this study was to develop a transgenic rice line with high-level accumulation of β-glucanase in seeds. A codon-optimized synthetic gene (gb:U33212) encoding this enzyme was transformed into rice (Oryza sativa L. ssp.japonica) under the control of the rice seed storage protein Gtl promoter. The transgenic line C19was identified as the one with the highest endoglucanase activity among the total of36independent transgenic lines obtained. The cellulase activity in the C19seeds was estimated at about830unit/g of dried seeds using CMC-Na as substrate.The enzymes produced in the seeds had an optimum pH of5.0 and optimum temperature of80℃, which is similar to the enzymes produced by the native bacterium host. The cellulase activity in the rice seeds was roughly equal among the seeds harvested from different generations, no significant activity was lost during the storage. This study demonstrates that the transgenic rice seeds could be used as a bioreactor for production of enzymes for cellulosic biomass conversion.Human Serum Albumin is widely used in vaccine formulation, cell culture media, drug delivery, in vivo diagnostics and etc.Transgenic rice seeds provide a commercially feasible heterologous expression system for recombinant Human Serum Albumin. It is the purpose of this study to develop a new downstream purification process aimed at non-clinical application to meet the increasing demand. The rHSA can be purified by a combination of specified steps in which an extraction supernatant obtained from rice seeds is subjected to heat treatment and acid/alkali sedimentation, followed by subsequent treatments with ANX FF anion exchange chromatography and Butyl HP hydrophobic chromatography, and by ultrafiltration to obtain a pure form of rHSA. The total recovery was62.4%and the product fulfils the purity criteria of the99%. The results obtained from MALDI-TOF mass spectrometry and N-terminal sequence determination suggest the purified rHSA identical to its natural counterpart. The entire downstream purification scheme is simple, reproducible and well suited for scaling-up to an industrial operation. |
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