Cell-free expression and functionality analysis of the tobacco lectin

Abstract

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.

Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells

“…The insect midgut cell line CF-203 that originated from the midgut of the eastern spruce budworm Choristoneura fumiferana (Palli et al, 1997), was cultured in Insect-Xpress medium (BioWhittaker-Cambrex Bioscience, Walkersville, MD) supplemented with 2.5% heat-inactivated fetal bovine serum (FBS) (Sigmae Aldrich, Bornem, Belgium) as reported before (Vandenborre et al, 2008).…”

Insect cell culture and applications to research and pest management

“…However, it should be emphasized that fine specificity of different lectins toward oligosaccharides and glycans can be very different even though they interact with the same monosaccharide. To complete these studies, tagged lectin was used to visualize lectin binding to membrane receptor(s) and cellular internalization (Vandenborre et al 2008). The use here of cultured midgut cells facilitates the investigation of the glycosylation process in insects and the interaction of lectin with receptor proteins.…”

Photorhabdus luminescensPirAB-fusion protein exhibits both cytotoxicity and insecticidal activity

“…The CF-203 cell line, originating from the midgut of the spruce budworm (Choristoneura fumiferana; Lepidoptera, Torticididae) was the kind gift of Prof. Guido F. Caputo (Natural Resource Canada). CF-203 was cultured in Insect-Xpress medium (BioWhittaker, Cambrex Bioscience, Walkersville, MD) supplemented with 2.5% heat-inactivated fetal bovine serum (Sigma-Aldrich, Bornem, Belgium) at 27°C ( Vandenborre et al, 2008).…”