Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector

Insertion in the coding region of the movement protein improves stability of the plasmid encoding... 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-γ) and successfully utilized to produce HuIFN-γ in both BY-2 protoplasts and transgenic BY-2 cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector

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Publisher
Springer Vienna
Copyright
Copyright © 2008 by Springer-Verlag
Subject
Biomedicine; Infectious Diseases; Medical Microbiology ; Virology
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-008-0165-z
Publisher site
See Article on Publisher Site

Abstract

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-γ) and successfully utilized to produce HuIFN-γ in both BY-2 protoplasts and transgenic BY-2 cells.

Journal

Archives of VirologySpringer Journals

Published: Sep 1, 2008

References

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