title: High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system.
abstract: Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8-4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid. The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53-39 mg/L per cycle during five recycling cycles.
authors.affiliation: Department of Molecular Biology, Institute of Molecular Biology and Genetics, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
authors.forename: Nan-Sun
authors.initials: NS
authors.lastname: Kim
authors.affiliation: Department of Molecular Biology, Institute of Molecular Biology and Genetics, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
authors.forename: Hwa-Young
authors.initials: HY
authors.lastname: Yu
authors.affiliation: Department of Molecular Biology, Institute of Molecular Biology and Genetics, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
authors.forename: Nguyen-Duc
authors.initials: ND
authors.lastname: Chung
authors.affiliation: Natural Bio-Materials Inc., 809-2 Yongam-ri, Bongdong-eup, wanju-gun, Jeollabuk-do, Republic of Korea.
authors.forename: Tae-Ho
authors.initials: TH
authors.lastname: Kwon
authors.affiliation: Department of Molecular Biology, Institute of Molecular Biology and Genetics, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea. Electronic address: mskyang@chonbuk.ac.kr.
authors.forename: Moon-Sik
authors.initials: MS
authors.lastname: Yang
mesh_terms: D000681:Amylases / Q000235:genetics; D000818:Animals; D002244:Carbon / Q000378:metabolism; D002417:Cattle; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003470:Culture Media; D005650:Fumarates / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D008293:Malates / Q000378:metabolism; D012275:Oryza / Q000166:cytology*; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019802:Succinic Acid / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D013535:Suspensions; D014357:Trypsin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003470:Culture Media; D005650:Fumarates; D008293:Malates; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013535:Suspensions; D013395:Sucrose; D002244:Carbon; C030298:malic acid; C032005:fumaric acid; D019802:Succinic Acid; D000681:Amylases; D014357:Trypsin
keywords: Alternative carbon source; Recombinant trypsin; Transgenic rice cell suspension culture