Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris

被引:30
作者
He, Wenqin [1 ,2 ,3 ,4 ]
Zhu, Yuanyuan [5 ]
Shirke, Abhijeet [1 ,2 ,3 ,4 ]
Sun, Xiaojun [6 ]
Liu, Jian [7 ]
Gross, Richard A. [1 ,2 ,3 ,4 ]
Koffas, Mattheos A. G. [1 ,2 ,3 ,4 ]
Linhardt, Robert J. [1 ,2 ,3 ,4 ]
Li, Ming [8 ]
机构
[1] Rensselaer Polytech Inst, Ctr Biotechnol & Interdisciplinary Studies, Dept Chem & Chem Biol, Troy, NY 12180 USA
[2] Rensselaer Polytech Inst, Dept Biol, Ctr Biotechnol & Interdisciplinary Studies, Troy, NY 12180 USA
[3] Rensselaer Polytech Inst, Ctr Biotechnol & Interdisciplinary Studies, Dept Chem & Biol Engn, Troy, NY 12180 USA
[4] Rensselaer Polytech Inst, Dept Biomed Engn, Ctr Biotechnol & Interdisciplinary Studies, Troy, NY 12180 USA
[5] Nanjing Forestry Univ, Dept Chem Proc Engn Forest Prod, Nanjing, Jiangsu, Peoples R China
[6] Shandong Univ, Natl Glycoengn Res Ctr, Shandong Prov Key Lab Carbohydrate Chem & Glycobi, State Key Lab Microbial Technol, Jinan 2500100, Shandong, Peoples R China
[7] Univ N Carolina, Eshelman Sch Pharm, Div Chem Biol & Med Chem, Chapel Hill, NC USA
[8] Tianjin Univ Sci & Technol, Coll Biotechnol, Key Lab Ind Fermentat Microbiol, Tianjin, Peoples R China
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
Chondroitin; Sulfotransferase; Bacterial expression; Yeast expression; Glycosylation; Kinetics; MOLECULAR-CLONING; CHONDROITIN SULFATE; HEPARAN-SULFATE; SULFOTRANSFERASES; BIOSYNTHESIS; 4-O-SULFOTRANSFERASES; ENZYMES; PRODUCE; FAMILY;
D O I
10.1007/s00253-017-8411-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.
引用
收藏
页码:6919 / 6928
页数:10
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