title: Production of Bioactive Recombinant Bovine Chymosin in Tobacco Plants.
abstract: Chymosin (also known as rennin) plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA). The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen.
authors.affiliation: Agronomy College, Shenyang Agricultural University, No. 120 Dongling Rd., Shenhe Distirct, Shenyang 110866, China. weizy@cjaas.com.|Laboratory of Plant Bioreactor and Genetics Engineering, Jilin Provincial Key Laboratory of Agricultural Biotechnology, Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, No. 1363 Shengtai St., Changchun 130033, China. weizy@cjaas.com.
authors.forename: Zheng-Yi
authors.initials: ZY
authors.lastname: Wei
authors.affiliation: Laboratory of Plant Bioreactor and Genetics Engineering, Jilin Provincial Key Laboratory of Agricultural Biotechnology, Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, No. 1363 Shengtai St., Changchun 130033, China. yuying0609@126.com.|State Key Labortory of Agrobiotechnology, China Agricultural University, No. 2 West Yuanmingyuan Rd., Beijing 100094, China. yuying0609@126.com.
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Zhang
authors.affiliation: Laboratory of Plant Bioreactor and Genetics Engineering, Jilin Provincial Key Laboratory of Agricultural Biotechnology, Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, No. 1363 Shengtai St., Changchun 130033, China. wangypbio@163.com.
authors.forename: Yun-Peng
authors.initials: YP
authors.lastname: Wang
authors.affiliation: Agronomy College, Shenyang Agricultural University, No. 120 Dongling Rd., Shenhe Distirct, Shenyang 110866, China. fan_mingxia@foxmail.com.
authors.forename: Ming-Xia
authors.initials: MX
authors.lastname: Fan
authors.affiliation: Laboratory of Plant Bioreactor and Genetics Engineering, Jilin Provincial Key Laboratory of Agricultural Biotechnology, Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, No. 1363 Shengtai St., Changchun 130033, China. zhongxf@cjaas.com.
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Zhong
authors.affiliation: Chashan Higher Education Zone, Wenzhou University, Wenzhou 325035, China. seer.sino@hotmail.com.
authors.forename: Nuo
authors.initials: N
authors.lastname: Xu
authors.affiliation: Agronomy College, Shenyang Agricultural University, No. 120 Dongling Rd., Shenhe Distirct, Shenyang 110866, China. fenglinsn@126.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Lin
authors.affiliation: Laboratory of Plant Bioreactor and Genetics Engineering, Jilin Provincial Key Laboratory of Agricultural Biotechnology, Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, No. 1363 Shengtai St., Changchun 130033, China. xingsc@cjaas.com.
authors.forename: Shao-Chen
authors.initials: SC
authors.lastname: Xing
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002417:Cattle; D017796:Caulimovirus / Q000235:genetics; D012085:Chymosin / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D012085:Chymosin
keywords: chymosin; milk coagulation; rennin; tobacco; transformation