Identification and Quantification of Endogenous Plant β-glucosidase in Transgenic Rice Cells

초록

U.S. FDA approved the plant-cell expressed recombinant form of acid β-glucosidase in 2012. It is also known as glucocerebrosidase (GCD), which is a hydrolytic lysosomal enzyme indicated for enzyme replacement therapy of Gaucher disease. In this study, GCD was expressed in transgenic rice (Oryza sativa) cells and its characteristics were investigated. GCD assay was performed by using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as a β-glucosidase substrate. In rice cells, several endogenous plant β-glucodisases are found and their activities interfere with the detection of target GCD. Real-time quantitative PCR was performed for the identification and relative quantification of gene expression of plant β-glucosidases (i.e., Os3bglu7 and Os3bglu8) in transgenic rice cells. As a result, expression of plant β-glucosidase genes was identified in various rice cell lines producing recombinant GCD and hCTLA4Ig as well as in wild-type rice cells. These results indicate that the existence of endogenous plant β-glucosidases is an obstacle against GCD assay. In conclusion, separation of target GCD among the mixtures including plant β-glucosidases is needed for correct GCD assay in transgenic rice cells.