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A method for separating and purifying recombinant human serum albumin (rHSA) from transgenic rice grain, sequentially comprising the steps of: 1) subjecting crude extract of rHSA to cation exchange chromatography to obtain primary product I; 2) subjecting the primary product I to anion exchange chromatography to obtain secondary product II; 3) subjecting the secondary product II to hydrophobic chromatography to obtain purified rHSA. The method may further comprise a step of ceramic hydroxyapatite chromatography prior to the hydrophobic chromatography. The method has the advantages of low cost and easy operation. The resultant rHSA has a purity of about 99% by HPLC.


Patent
Healthgen Biotechnology Corporation | Date: 2016-01-14

A method for extracting recombinant human serum albumin (rHSA) from transgenic rice grain is provided, comprising the steps of: 1) grinding dehusked rice containing rHSA into milled rice grain with a fineness of 80120 mesh, which is mixed with a extraction buffer in a wily ratio of 1:5, then extracting at 5560 C. for 13 hours to obtain mixture 1; said extraction buffer comprises 1030 mM phosphate buffer, 1020 mM sodium acetate, 1530 mM ammonium sulfate and 520 mM sodium caprylate and has a pH of 6.58; 2) adjusting the pH of mixture I to 4.04.5, followed by precipitating at room temperature for 312 hours to obtain mixture 3) filtering the mixture 11 and collecting the filtrate, to obtain a solution containing high concentration of rHSA. The concentration of rHSA in the resultant solution is 650660 g/mL, which increases by 1.15 times comparing to the extraction amount before improvement, and the amount of non-target protein is reduced by 2.46 times. The method provides a basis for subsequent purification of rHSA.


A method for separating and purifying recombinant human serum albumin (rHSA) from transgenic rice grain, sequentially comprising the steps of: 1) subjecting crude extract of rHSA to cation exchange chromatography to obtain primary product I; 2) subjecting the primary product Ito anion exchange chromatography to obtain secondary product II; 3) subjecting the secondary product II to hydrophobic chromatography to obtain purified rHSA. The method may further comprise a step of ceramic hydroxyapatite chromatography prior to the hydrophobic chromatography. The method has the advantages of low cost and easy operation. The resultant rHSA has a purity of about 99% by HPLC.


Patent
Healthgen Biotechnology Co. | Date: 2011-08-10

A method for extracting recombinant human serum albumin (rHSA) from transgenic rice grain is provided, comprising the steps of: 1) grinding dehusked rice containing rHSA into milled rice grain with a fineness of 80120 mesh, which is mixed with a extraction buffer in a w/v ratio of 1:5, then extracting at 5560 C. for 13 hours to obtain mixture I; said extraction buffer comprises 1030 mM phosphate buffer, 1020 mM sodium acetate, 1530 mM ammonium sulfate and 520 mM sodium caprylate and has a pH of 6.58; 2) adjusting the pH of mixture I to 4.04.5, followed by precipitating at room temperature for 312 hours to obtain mixture II; 3) filtering the mixture II and collecting the filtrate, to obtain a solution containing high concentration of rHSA. The concentration of rHSA in the resultant solution is 650660 g/mL, which increases by 1.15 times comparing to the extraction amount before improvement, and the amount of non-target protein is reduced by 2.46 times. The method provides a basis for subsequent purification of rHSA.


Zhang L.,Rice University | Shi J.,Healthgen Biotechnology Ltd. Co. | Jiang D.,Healthgen Biotechnology Ltd. Co. | Stupak J.,National Research Council Canada | And 5 more authors.
Journal of Biotechnology | Year: 2013

Human alpha-antitrypsin (AAT) is the most abundant circulating protease inhibitor in the human plasma. It is produced in the liver and exerts a primary physiological role as inhibitor for the neutrophil elastase in the lung. Individuals with one or several gene mutations in AAT causing reduction of the protein are related to lung, liver and pancreatic emphysema diseases and are treated lifelong with infusions of human plasma-derived AAT. Due to shortage of plasma and low expression levels of recombinant AAT in conventional gene expression systems, we explored the possibility to produce recombinant AAT in rice grains (Oryza sativa AAT, OsrAAT). An expression level of up to 2.24. g/kg brown rice and a final recovery of purified 0.366. g/kg OsrAAT has been obtained. OsrAAT has the same secondary structure and protease inhibitory activity as plasma-derived AAT (pAAT), but was highly heterogeneous with regard to glycan modifications. Thus 32.8% of OsrAAT were glycosylated and 67.2% were free of glycans as determined by MALDI-MS. Of the N-glycan structures 64.8% were vacuole-specific paucimannosidic molecules. Immune electron microscopy located OsrAAT in the endoplasmic reticulum lumen as precursor-accumulating (PAC)-like vesicle structures. The pharmacokinetic study indicated that the half-life of OsrAAT was prolonged, while the clearance rate was faster than that of pAAT in vivo. The results demonstrate that rice endosperm is a promising system to express this biopharmaceutical protein. © 2013 Elsevier B.V. Source

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