Garlic (Allium sativum) belongs to the Amarillidaceae family and has several lectins in roots (ASAr), leaves (ASAL) and bulbs (ASAI and ASAII). In this work, we extracted and purified lectins from garlic bulbs by fractionated precipitation using ammonium sulphate (30%s-80%s) and ionic interchange chromatography, purifying both isolectins (ASAI and ASAII) together, due to their similar properties (molecular weight, isoelctric point). ASAI is a 11,5 KDa and 12,5 kDa heterodimer and ASAII is a 24 kDa homodimer (two identical 12 kDa subunits). Both lectins recognize mannose, but come from different genes and are also differents in glycosylation patterns: ASAI precursor is monoglycosylated, not the mature form and ASAII is not glycosylated in any form. After the purification process, one only band around 12 kDa was obtained (SDS-PAGE electrophoresis), with agglutination activity in rabbit red blood cells. In addition, total RNA was extracted (Trizol method), used in RT-PCR to get the cDNA of ASAII gene, amplified and cloned in E. coli cultures. The 330 bp gene produced a recombinant protein of about 12 kDa which was purified, renatured and solubilized in PBS buffer. This protein has similar properties to ASAII lectin and has agglutinant activity in rabbit red blood cells. This is a novel methodology to produce this lectin in an active form.
[1] Carreño, N. Evaluación de la actividad insecticida de lectinas de bulbo de ajo. MSc graduation Thesis. University National of Colombia. Unpublished.
[2] Fitches, E., Wiles, D., Douglas, A., Hinchliffe, G., Audsley, Neil., Gatehouse, J. (2008). The insecticidal activity of recombinant garlic lectins toward aphids. Insect Biochem and Mol Biology. 38: 905-915.
