Production and Purification of Recombinant Glucocerebrosidase in Transgenic Rice Cell Suspension Cultures.
Gaucher disease, which is caused by deficiency of glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy. Plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. However, the gliding step for the purification is essential since the produced protein still exists inside cells. In the case of rice-amylase 1A (RAmy1A) secretion signal peptide, GCD protein is secreted outside of cells and simplifies the purification step. Here, an established cell line was confirmed as having fundamental characteristics of growth and production. GCD from transgenic calli was examined by Western blot analysis and compared with that from Chinese hamster ovary (CHO) cells. Calli expressing high levels of GCD were used to establish suspension cell lines. Growth and production characteristics were investigated in suspension cell cultures. Production of GCD in suspension cultures was confirmed upon induction for 12-24 h. The amount of GCD in medium increased until 60-84 h and decreased thereafter. Purification of GCD was performed in three steps (ion exchange, hydrophobic interaction, and size exclusion chromatography) and verified. Purified GCD was able to hydrolyze the synthetic substrate. Thus, a rice expression system could be a suitable alternative to GCD expression in mammalian cells.
Department of Biological Engineering, Inha University, Incheon, 402-751, South Korea.
Hyung-Jin
HJ
Nam
Department of Biological Engineering, Inha University, Incheon, 402-751, South Korea.
Jun-Young
JY
Kwon
Department of Biological Engineering, Inha University, Incheon, 402-751, South Korea.
Hong-Yeol
HY
Choi
Boryung Pharmaceutical Co., Ansan, Gyeonggi, 425-120, South Korea.
Seung-Hoon
SH
Kang
Boryung Pharmaceutical Co., Ansan, Gyeonggi, 425-120, South Korea.
Hahn-Sun
HS
Jung
Department of Biological Engineering, Inha University, Incheon, 402-751, South Korea. kimdi@inha.ac.kr.
Dong-Il
http://orcid.org/0000-0001-7633-382X
DI
Kim
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D049109:Cell Proliferation; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D005962:Glucosylceramidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012275:Oryza / Q000166:cytology* / Q000235:genetics*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D013535:Suspensions; D000516:alpha-Amylases / Q000235:genetics
D011994:Recombinant Proteins; D013535:Suspensions; D000516:alpha-Amylases; D005962:Glucosylceramidase
Gaucher disease; Glucocereborosidase (GCD); RAmy3D promoter; Transgenic rice cell cultures