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MSc. by Research in Crop Improvement by Molecular Biotechnology (2005)

A research degree based on a (one year) research project on crop improvement by molecular biotechnology at Coventry University:

  Characterisation and heterologous expression of Agaricus bisporus lectin which inhibits epithelial cell proliferation

The lectin from the common edible mushroom Agaricus bisporus specifically recognizes the Thomsen-Friedenreich (TF) - antigen (galactose-β-1, 3-N-acetylgalactosamine-α), sialyl-2, 3-galactosyl-β-1, 3-N-acetyl-galactosamine-α and N-acetylglucosamine. The anti-cancer properties of the A. bisporus lectin are well documented, using purified mushroom extracts which contain a mix of five different isoforms of the lectin (Carrizo et al., 2005). Purified samples of each isoform are required to perform further cell-line based clinical studies. Previous work at Warwick HRI has identified two allelic version of the lectin gene from A. bisporus. During this project, it became clear that these two allelic versions are two different genes, abl1 and abl2. Also, another allelic version of abl1 was identified. The lectin gene abl1 was amplified, cloned in the pET-15b bacterial expression vector, expressed in E. coli, extracted from the bacterial cells and purified using immobilised metal affinity chromatography. Heterologously expressed ABL1 accumulated in the insoluble protein cell extract fraction when the bacterial cultures were grown at 37°C. Homology studies with other proteins had suggested that the lectin would be soluble. A literature search suggested accumulation of the heterologously expressed protein in inclusion bodies because of improper folding due to high protein production rates at high incubation temperatures. This hypothesis was confirmed by incubating the bacterial cultures at lower temperatures after which analysis showed accumulation of lectin in the soluble fraction. The most efficient procedure was incubation of the bacterial cultures at 20°C after induction of protein production. The purification procedure required optimisation for scaling-up of protein production in 1l cultures. Once these procedures were optimised, the same techniques were used for the expression of the second lectin gene, abl2. The purified white button mushroom lectin isoforms, ABL1 and ABL2S, were then concentrated and their activity was investigated by using a blood agglutination assay. Both ABL1 and ABL2S have shown clear ability to agglutinate red blood cells and are therefore active.This is the first report of active, heterologously expressed and purified A. bisporus lectin. This study optimised the production of pure, single isoform lectin in thigh quantities which can be used for experiments on inhibition of epithelial cancer cell growth.The procedures optimised for lectin expression, were also used for the expression of another novel mushroom gene, cbp1 (a putative cruciform binding protein).

 

 blood1.jpg

Agglutination test assessing the functionality of the purified mushroom lectin (left tube) compared to normal blood (right tube), both samples are heparin treated to prevent natural agglutination. Active lectin will agglutinate human (my own actually) red blood cells.

 

Futher information on this project can not be given in regard to future patent acquisition

Supervisors: D. Eastwood1, S. Sreenivasaprasad1, K. Burton1 and J. Henderson2

1. Warwick HRI Wellesbourne, Warwick, UK

2. Coventry University, Coventry, UK