Cell-free expression and functionality analysis of the tobacco lectin.
The Nicotiana tabacum lectin, called Nictaba, is a nucleocytoplasmic plant lectin expressed in tobacco leaves after exogenous application of specific jasmonates and upon insect herbivory. Since the lectin concentrations are rather low, huge amounts of plant material are needed to purify milligram quantities of the protein. In addition, the purified lectin fractions are always contaminated with low molecular weight compounds such as phenols. In an attempt to improve and facilitate the purification of the tobacco lectin in reasonable amounts, an in vitro-coupled transcription/translation system based on an Escherichia coli lysate was used to express the lectin gene. Recombinant expression levels could be enhanced by an adapted codon usage. Recombinant lectin was purified, biochemically characterized and found to be biologically active. The biological activity of the recombinant lectin towards insect epithelial midgut cells was clearly demonstrated in a functional bio-assay and the internal cellular localization was analyzed using immunocytochemical techniques.
Vandenborre
Gianni
G
Department of Molecular Biotechnology, Lab. Biochemistry and Glycobiology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Lannoo
Nausicaä
N
Smagghe
Guy
G
Daniel
Erica
E
Breite
Andrew
A
Soin
Thomas
T
Jacobsen
Linda
L
Van Damme
Els J M
EJ
D000371:Agglutination / Q000187:drug effects; D000818:Animals; D015153:Blotting, Western; D050260:Carbohydrate Metabolism / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D041981:Gastrointestinal Tract / Q000378:metabolism; D007313:Insecta / Q000166:cytology / Q000187:drug effects; D037102:Lectins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013058:Mass Spectrometry; D014176:Protein Biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014026:Nicotiana / Q000378:metabolism*
D037102:Lectins; D011994:Recombinant Proteins