title: Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do 561-756, Republic of Korea; Department of Bioactive Material Science, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
authors.forename: Jae-Wan
authors.initials: JW
authors.lastname: Jung
authors.affiliation: Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
authors.forename: Nan-Sun
authors.initials: NS
authors.lastname: Kim
authors.affiliation: Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
authors.forename: Seon-Hui
authors.initials: SH
authors.lastname: Jang
authors.affiliation: Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
authors.forename: Yun-Ji
authors.initials: YJ
authors.lastname: Shin
authors.affiliation: Department of Molecular Biology, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do 561-756, Republic of Korea; Department of Bioactive Material Science, Chonbuk National University, 664-14 Dukjindong, Jeonju, Jeollabuk-do 561-756, Republic of Korea. Electronic address: mskyang@jbnu.ac.kr.
authors.forename: Moon-Sik
authors.initials: MS
authors.lastname: Yang
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000378:metabolism; D018745:Genome, Plant; D006801:Humans; D012275:Oryza / Q000235:genetics*; D059828:Plant Cells / Q000378:metabolism*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013535:Suspensions; D013997:Time Factors; D014170:Transformation, Genetic; D000520:alpha-Glucosidases / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D018744:DNA, Plant; D011134:Polysaccharides; D011994:Recombinant Proteins; D013535:Suspensions; C509951:GAA protein, human; D000520:alpha-Glucosidases
keywords: Acid α-glucosidase (GAA); Agrobacterium tumefaciens; Pompe disease; Rice α-amylase 3D (RAmy3D) promoter; Transgenic rice cell suspension culture