Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector.
Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.
Kumagai
M H
MH
Biosource Technologies, Vacaville, CA, USA. monto@davis.com
Donson
J
J
della-Cioppa
G
G
Grill
L K
LK
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006031:Glycosylation; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics* / Q000821:virology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017898:Tobamovirus / Q000235:genetics* / Q000648:ultrastructure; D014162:Transfection; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases