Production and purification of active snowdrop lectin in Escherichia coli

Abstract

Recombinant snowdrop lectin was produced in Escherichia coli from a cDNA clone encoding mature Galanthus nivalis agglutinin. After induction with isopropylthio-β-D-galactoside, inclusion bodies from E. coli were solubilised and the G. nivalis agglutinin purified by metal-affinity chromatography using a carboxy-terminal hexahistidine tag. The protein was refolded on the metal-affinity column prior to elution. After purification, the recombinant G. nivalis agglutinin agglutinated rabbit erythrocytes to a dilution similar to that determined for 'native' lectin purified from snowdrop, and showed similar specific binding to mannose. The toxicity of the recombinant G. nivalis agglutinin towards rice brown planthopper (Nilaparvata lugens) was shown to be similar to that of 'native' G. nivalis agglutinin when incorporated into an artificial diet. The recombinant G. nivalis agglutinin is thus functionally similar to 'native' snowdrop lectin.Keywords : snowdrop (Galanthus nivalis); lectin ; bacterial expression system; recombinant protein; functional activity.Plant lectins constitute a heterogeneous group of proteins eration of Escherichia coli in the small intestine of rats when with different biochemical properties and carbohydrate-binding included as part of the diet [10]. specificities. They are widespread throughout the plant kingdom,In its natural state, the snowdrop lectin isolated from bulbs occurring in a number of plant species from all major taxonomic exists as a highly complex mixture of isoforms as evidenced by groupings. Lectins found in plants of the monocotyledonous isoelectric focussing [4]. Since snowdrop bulb tissue is rich in group are distinct from those of the dicotyledons and exhibit polysaccharides, which interfere with extraction processes, several unique properties. Snowdrop lectin (Galanthus nivalis young developing ovaries, which contain high concentrations of agglutinin) was first isolated from bulbs of the snowdrop G. lectin and are low in RNAse activity, were chosen as the starting nivalis L., and characterised as a tetrameric protein of 50 kDa material for nucleic acid extraction [11]. A cDNA library was composed of four identical subunits [1]. This lectin is unique in constructed from total poly(A)-rich RNA isolated from snowthat it recognizes A-D-mannose groups, in particular those link-drop ovaries and used to obtain five lectin clones [11], which ages made by Man(A1-3)Man units [2]; this exclusive specificity differed with respect to their nucleotide sequence and deduced for mannose is shared by members of the families Amaryllida-amino acid sequence. One of these clones, LECGNA2, was subceae [3] and Alliaceae [4] and distinguishes snowdrop lectin sequently used to produce constructs for transformation of the from the mannose/glucose-binding lectins of legumes such as G. nivalis agglutinin gene into plants [12Ϫ14]. jackbean (Canavalia ensiformis L. DC) and pea (Pisum sativum A number of plant lectins, including snowdrop lectin, have L.). Crystallographic studies have confirmed that G. n...

The Phytochemical Constitution of Maltese Medicinal Plants – Propagation, Isolation and Pharmacological Testing

“…The agglutination was quantified by lysing the precipitated agglutinated cells and read spectrophotometrically at a wavelength of 405 nm at 20, 40, 60 and 80 minutes. Over the 80-min period, the best results were obtained after 60 min, as was observed by [170] for the snowdrop lectin. Extracts from all three plant parts exhibited superior haemagglutination activity (AgA) to the standard PHA lectin (AgA -3.996 ± 0.259).…”

Cloning, expression and functional characterisation of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase

“…The sequence coding for GNA in chitinase/GNA omitted the C-terminal extension removed from the protein in planta; this truncated form of GNA has been shown to be fully active as a lectin (Longstaff et al, 1998). The predicted protein products were arranged in frame with the yeast a-factor N-terminal secretory signal.…”

Cloning, Overexpression, Purification, and Matrix-Assisted Refolding of DevS (Rv 3132c) Histidine Protein Kinase of Mycobacterium tuberculosis

“…Recent advances in protein folding have led to the development of matrix-bound renaturation techniques which could be very effective in folding proteins not amenable to conventional refolding protocols (7,8). Solubilization of proteins on a solid matrix was previously reported by many groups on a variety of matrices including heparin-Sepharose, cellulose (20), and Ni2+-NTA, among others (10,(17)(18)(19). Ni2+-NTA resin is a matrix of choice since it is based on the specific affinity of Ni2+-NTA to 6× His and allows conformation-independent binding of tagged proteins.…”

“…ing overcomes this problem, as the matrix-bound protein is prevented from interacting with surrounding ~ 4oooo protein molecules and the d e n a t u r a n t can be completely ~= removed, thereby preventing aggregation (17)(18)(19)(20). We 20000 have overexpressed and refolded histidine sensor kinase DevS of M. tuberculosis using a matrix-assisted o refolding protocol.…”