Healthgen Biotechnology Ltd. Co.

Wuhan, China

Healthgen Biotechnology Ltd. Co.

Wuhan, China
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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.


An N.,Rice University | Ou J.,Wuhan Institute of Biotechnology | Ou J.,Healthgen Biotechnology Ltd. Co. | Jiang D.,Wuhan Institute of Biotechnology | And 9 more authors.
International Journal of Molecular Sciences | Year: 2013

Basic fibroblast growth factor (FGF-2) is an important member of the FGF gene family. It is widely used in clinical applications for scald and wound healing in order to stimulate cell proliferation. Further it is applied for inhibiting stem cell differentiation in cultures. Due to a shortage of plasma and low expression levels of recombinant rbFGF in conventional gene expression systems, we explored the production of recombinant rbFGF in rice grains (Oryza sativa bFGF, OsrbFGF). An expression level of up to 185.66 mg/kg in brown rice was obtained. A simple purification protocol was established with final recovery of 4.49% and resulting in a yield of OsrbFGF reaching up to 8.33 mg/kg OsrbFGF. The functional assay of OsrbFGF indicated that the stimulating cell proliferation activity on NIH/3T3 was the same as with commercialized rbFGF. Wound healing in vivo of OsrbFGF is equivalent to commercialized rbFGF. Our results indicate that rice endosperm is capable of expressing small molecular mass proteins, such as bFGF. This again demonstrates that rice endosperm is a promising system to express various biopharmaceutical proteins. © 2013 by the authors; licensee MDPI, Basel, Switzerland.


PubMed | Healthgen Biotechnology Corporation and Wuhan University
Type: | Journal: Journal of biotechnology | Year: 2016

Rice seed is a cost-effective bioreactor for the large-scale production of pharmaceuticals. However, convincing evidence of the immunogenicity of plant-specific glycans is still limited although plant-specific glycans are considered potential allergic antigens. In the present study, we found that the -1,3-fucose content of the glycoprotein produced from rice seed was much lower than that in leaf, and conversely, a higher -1,2-xylose content was detected in seed than that in leaf. We detected the -1,6-fucose content in the glutelin and recombinant human 1-antitrypsin (OsrAAT). The further results in a line containing AAT and FUT8 genes indicated that the -1,6-fucose content of modified glycosylated recombinant 1-antitrypsin (mgOsrAAT) was 38.4%, while glutelin was only 6.8%. Interestingly, the -1,3-fucose content of mgOsrAAT was significantly reduced by 59.8% compared with that of OsrAAT. Furthermore, we assessed the immunogenicity of OsrAAT, mgOsrAAT and human 1-antitrypsin (hAAT) using an animal system. The PCA results indicated no significant differences in the IgG, IgM and IgE titers among OsrAAT, mgOsrAAT and hAAT. Further studies revealed that those antibodies were mainly from -1,3-fucose, but not from -1,2-xylose, indicating that -1,3-fucose was the major immunogenic resource. Our results demonstrated that -1,3-fucose contents in seed proteins was much less than that of leaf, and could not be a plant-specific glycan because it also exists in human proteins.


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.


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 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.

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