Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector.
A major obstacle in the genetic manipulation of tomato mosaic virus (ToMV) is the instability of the plasmid containing the infectious full-length cDNA of the ToMV vector, which often prevents the subcloning of a foreign gene of interest into the vector. We found that an insertion of a 0.3-1.6-kbp DNA fragment in the movement protein (MP) coding region effectively attenuated bacterial toxicity of the plasmid and greatly increased plasmid yield. Accumulation of a modified ToMV containing a 0.3-kb insertion in the MP coding region was comparable to that of a modified ToMV without an insertion in tobacco BY-2 protoplasts, while an insertion more than 0.6 kb significantly reduced accumulation of the viral RNA. The modified ToMV vector containing a 0.3-kb insertion was easily manipulated to introduce a coding sequence for human interferon-gamma (HuIFN-gamma) and successfully utilized to produce HuIFN-gamma in both BY-2 protoplasts and transgenic BY-2 cells.
Dohi
Koji
K
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan.
Tamai
Atsushi
A
Mori
Masashi
M
D002460:Cell Line; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D053830:Plant Viral Movement Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D014026:Nicotiana / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000378:metabolism
D053830:Plant Viral Movement Proteins; D007371:Interferon-gamma