Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture

• Published in: Journal of biotechnology 2016-05, Vol.226, p.44-53
• Main Authors: Jung, Jae-Wan, Kim, Nan-Sun, Jang, Seon-Hui, Shin, Yun-Ji, Yang, Moon-Sik
• Format: Article
• Language: English
• Subjects: Acid α-glucosidase (GAA); Agrobacterium tumefaciens; alpha-Glucosidases - biosynthesis; alpha-Glucosidases - isolation & purification; Base Sequence; Blotting, Northern; Blotting, Western; Cell Culture Techniques - methods; Cloning, Molecular; Culture; DNA, Plant - genetics; Electrophoresis, Polyacrylamide Gel; Enzymes; Gene expression; Genetic Vectors - metabolism; Genome, Plant; Human; Humans; Mathematical analysis; Oryza - genetics; Oryza sativa; Plant Cells - metabolism; Plants, Genetically Modified; Polymerase Chain Reaction; Polysaccharides - metabolism; Pompe disease; Recombinant; Recombinant Proteins - biosynthesis; Rice α-amylase 3D (RAmy3D) promoter; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Suspensions; Time Factors; Transformation, Genetic; Transgenic; Transgenic rice cell suspension culture; Vectors (mathematics)
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2016.03.031

•We constructed transgenic rice cell suspension culture producing human GAA.•The expression of rhGAA was determined by Northern and western blot analyses.•The accumulated rhGAA protein in the culture medium was 37mg/L.•The rhGAA was purified using a Ni-NTA column.•The acid alpha-glucosidase activity of hGAA produced in transgenic rice cells gave results similar to those of the enzyme produced by CHO cells. Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid α-glucosidase (GAA), a glycogen-degrading lysosomal enzyme. In this study, the human GAA cDNA gene was synthesized from human placenta cells and cloned into a plant expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. Genomic DNA PCR and Northern blot analysis were used to determine the integration and mRNA expression of the hGAA gene in the putative transgenic rice cells. SDS-PAGE and Western blot analysis showed that the glycosylated precursor recombinant hGAA had a molecular mass of 110kDa due to the presence of seven N-glycosylation sites. The accumulation of hGAA protein in the culture medium was approximately 37mg/L after 11 days of culturing in a sugar depletion medium. The His tagged-hGAA protein was purified using an Ni-NTA column and confirmed as the precursor form of hGAA without the signal peptide encoded by the cDNA on the N-terminal amino acid sequence. The acid alpha-glucosidase activity of hGAA produced in transgenic rice cells gave results similar to those of the enzyme produced by CHO cells.