Isolation of a Galactose Specific Lectin from Moringa Oleifera Seeds: Affinity Purification and Characterization
by Kiran Kumar Tejavath, Sampurna Atmakuri & Siva Kumar Nadimpalli,
Laboratory of Protein Biochemistry and Molecular biology, University of Hyderabad, India.
A galactose-specific lectin was isolated from the crude extracts of the Moringa oleifera seed powder using a combination of ammonium sulphate fractionation (0-75%), Sepharose-galactose affinity chromatography and gel filtration on Sephadex G-150. Moringa oleifera seed lectin agglutinates pronase treated rabbit erythrocytes. It is a non-glycoprotein whose activity is inhibited by galactose and its derivatives. Additionally, sugar inhibition studies using synthetic sugars revealed some insights of the lectin properties. Galactosyl Naphthyl Imine, a galactose derivative was found to be a most potent inhibitor for Hemagglutinating activity (MIC= 0.04 mM). The native molecular mass of the purified lectin by MALDI-TOF analysis and gel filtration was found to be 25.8 kDa and 27.0 kDa, respectively. By SDS-PAGE analysis under reducing conditions, the protein gave a single band (Mr 27kDa) suggesting it to be a monomer. Interestingly the protein cross-reacts with an antibody to a galactose specific legume lectin (Dolichos lablab 31 kDa subunit antibody) suggesting some antigenic similarities among this non-legume and legume lectin, which share similar sugar specificity.
Lectins, by virtue of their exquisite sugar specificities and cell-agglutinating properties, have been found as useful tools in widespread applications for monitoring the expression of cell-surface carbohydrates as well as for the purification and characterization of glycoconjugates [1–3]. Extensive study of sequence homology and 3D structure of various plant lectins suggests that they are conserved throughout evolution and thus may play, yet unknown, important physiological roles [4, 5]. Seeds of legumes such as Dolichos lablab (Indian lablab beans), peas and beans have long been known to represent a rich source of lectins [6-8] in view of their high potential as tools in biochemical and biomedical research, both for preparative and for analytical purposes . We are looking for new lectins in the tropical and subtropical flora that might have special properties in terms of sugar-binding, specificity and /or stability. Moringa oleifera (MO) (family; Moringaceae) is a tropical plant having many medicinal values which is largely grown in India . Here we describe the affinity purification of a galactose specific lectin from the seeds of deshelled MO. The seeds contain high amounts of coagulant protein, which has been used to purify water and has also been characterized in our laboratory [11, 12]. There are reports on the isolation and identification of lectins from the MO seeds which differ in their properties from the galactose specific lectin we purified in this study [13-15].
Figure 1: Elution profile of the lectin from the affinity gel (3x15 cm): The ammonium sulphate fraction (70%) of Moringa oleifera saline extract was passed through the gel equilibrated with 0.15M NaCl. After washing the gel with 0.15M NaCl, extensively, the bound lectin was eluted using 0.3 M galactose in 0.15 M NaCl. Fractions of 1.5 mL were collected manually, protein was monitored at 280 nm.
The present work deals with the preparation of affinity matrix (Seralose-divinyl sulfone-galactose) which was successfully used by us for the purification of galactose-specific lectins from Dolichos lablab seeds . In the present study this matrix was used for the purification of the Moringa oleifera seed lectin (MoSL) that enabled us to study the properties of the lectin.
Materials : Dry mature seeds of Moringa oleifera (MO) PKM 1 variety were purchased locally. Synthetic sugars were kindly provided by Prof. C.P. Rao, IIT Mumbai. Seralose 6B, Sephadex G-150, divinyl sulfone, other chemical modification reagents used in the present study were of high purity and obtained from reputed firms.
Figure 2: Estimation of molecular weight using G-150 column (75x1.3cm): The column was calibrated with known standard proteins; i.e., BSA (66 kDa), Ovalbumin (45 kDa), Lysozyme (14.7 kDa), respectively. Arrow indicates fraction at which purified sample (Mr 27 kDa) was eluted. 1.0 mL fractions were collected. Inset is standard graph.
Preparation of Seralose-galactose affinity gel : Seralose-galactose affinity gel was prepared by activating 5 g of Seralose -6B with divinyl sulfone as described by Latha et al., , other researchers have also prepared the Sepharose–galactose gel following this protocol and used it effectively to purify galactose-specific lectins .
Extraction and purification of the lectin : All operations were done at 4 °C. Freshly dehulled Moringa oleifera seeds were finely ground into a fine powder. The flour was defatted using acetone and air dried at room temperature. The powder obtained was extracted at 4 °C with 0.15 M NaCl for 16 h. The crude slurry was centrifuged at 12000 rpm for 25 min. (NH4)2SO4 was added to the supernatant to attain 75% saturation, stirred for 2h. The precipitate was collected by centrifugation (12000 rpm, 25 min), dissolved in a small amount of saline, and dialysed extensively against saline. The dialysed sample was centrifuged as above and the clear supernatant was subjected to affinity chromatography.
Figure 3: 10% SDS-PAGE analysis of the purified lectin. Lane M: - Medium range molecular weight marker. Lane 2: - Seralosegalactose affinity purified MoSL under reducing conditions. The arrow corresponds to purified lectin. (Coomassie stain)
Affinity chromatography : Seralose-galactose gel was packed into a glass column and equilibrated with 0.15 M NaCl. The protein sample obtained above was then applied on the gel. The gel was washed extensively to remove unbound proteins and the bound protein eluted using 0.3 M galactose in 0.15 M NaCl. Protein was monitored at 280 nm. Protein containing fractions were dialyzed and tested for hemagglutinating activity using pronase treated rabbit erythrocytes. The active fractions were pooled, concentrated and subjected to gel filtration on Sephadex G-150 at a flow rate of 20 mL/h. The absorbance of the column fractions was monitored at 280 nm.