THE PATENTS ACT, 1970
PROVISIONAL SPECIFICATION
Section 10
"Recombinant Lectin and Method of Preparing thereof."
Unichem Laboratories Ltd., an Indian Company of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India.
And
Karnatak University, Dharwad 580 003, Karnatak, India.
The following specification particularly describes the nature of this invention:

FIELD OF INVENTION
The invention relates to Lectins, more particularly recombinant fungal lectins
BACKGROUND OF INVENTION
Lectins are proteins, or glycoproteins that agglutinate erythrocytes of some or all blood groups in vitro. They are important group of bioactive proteins found in most organisms. Lectins are used as tools for diagnostic and therapeutic purpose in health care areas. Lectins are also used in the purification of glycoproteins, oligosaccharide analysis and in cell selection processes. Lectins can bind reversibly with monosaccharides or the sugar moiety found in polysaccharides, glycoproteins or glycolipids.
Lectins are currently being considered for use as cancer therapeutic agents. Some lectins .have been shown to bind preferentially to cancer cell membranes and cause inhibition of cell growth. There are also reports of inhibition of angiogenesis. Lectins could inhibit cell adhesion, proliferation and colony formation. Some lectins modulate the growth, through proliferation or apoptosis of premalignant and malignant cells both in vitro and in vivo. Most of these effects are mediated by specific cell surface receptors. Several lectins show anti tumor activity. Because of their selective cell specificity lectins may serve as carriers for targeted drug delivery.
Several lectins have been isolated and characterized from a variety of sources. They however differ in their physico-chemical properties such as molecular size and sugar specificities. There is also characterization of a few recombinant proteins. The recombinant mushroom lectin from Marasmius


oreades has a molecular size of 33 kDa and shows high affinity for Galal, 3Gal and Galal, 3GalbM, 4GlcNAc. (R.P.Kruger et al., J.Biol.Chem., 277, 15002-15005, 2002; H.C.Winter et al. J.Biol.Chem ,277, 14996-15001, 2002., I.J. Goldstein, et al. US Patent :6958321)
A recombinant peanut lectin that shows affinity to Thomsen-Friedenreich antigen has also been cloned (V.Sharma and A.Surolia, Gene (Amst), 148, 299-304, 1994) and subsequently some mutants of this protein with a subunit molecular mass of ~28kDa has also been expressed in E.coli (V.Sharma, M.Vijayan and A.Surolia, J. Biol. Chem., 271, 21200-21213,1996) with some difference in its preference for sugar specificity. A much smaller lectin of molecular mass of 11.73 kDa with high binding affinity to L-fucose from Pseudomonas aeruginosa has also been expressed in E.coli as a Yellow fluorescent Protein-lectin fusion (BioTechniques, 41, 327-332, 2006).
SUMMARY OF THE INVENTION
The present invention provides a method for preparing a recombinant lectin expressed in E.coli. This method comprises the synthesis of a lectin gene sequence derived from the MADI MS/MS and X-ray crystallographic data of the Sclerotium rolfsii lectin, a soil borne phytopathogenic fungus, and its cloning in E.coli.
The recombinant lectin expressed as a soluble protein is purified by ion exchange and gel filtration chromatographic techniques.


The present invention provides a recombinant lectin that is specific for blood group Al cell and has sugar specificity towards GalNacal-3 (Fucal-2) Galbl-R also N-acetyl galactosamine Tn antigen but not towards TF antigen.
Further, the present invention provides a lectin that binds to human colon cancer, ovarian cancer and leukemic cells.
In one aspect this recombinant lectin in a biotinylated form or modified with a fluorogenic chromophore can be used for the detection of cancer cells or cancer associated specific antigens.
Its growth modulatory effect can have potential application in cancer therapy
In another aspect it can be used as a drug delivery agent for cancer treatment.
The invention discloses a DNA sequence coding for a 141 amino acids long lectin similar to the lectin (S.rolfsii), but with a few modifications was chemically synthesized and cloned into E.coli (SEQ ID 2 and 3). The cloned lectin was expressed after induction with the inducer IPTG. The expressed protein appeared in soluble and active form. The recombinant lectin was purified to essential homogeneity by resorting to anion exchange and molecular sieve chromatography. The mass spectrometric estimate of molecular weight show a value of 16.1 kDa. The isoelectric focusing experiment show the PI value close to 5.0.

The recombinant lectin exhibits specificity towards blood group Al and can be used for blood typing. The lectin binds specifically to human breast, colon and ovarian cancer tissues and distinct from other TF antigen binding lectins. Hence the lectin has application in cancer diagnosis. The lectin exhibits substrate specificity towards N-acetyl galactosamine, mucin and feutin. The lectin can be immobilized on a matrix and used to affinity purify glycoproteins and glycoconjugates that bind to this recombinant lectin.
This recombinant lectin can be used for the detection of certain glycoproteins and glycoconjugates present in biological fluids. This lectin can be used as a carrier of drugs to target certain cancer cells. The lectin may also find application as a diagnostic tool in the detection of microbial / viral pathogens.
DETAILED DESCRIPTION OF THE INVENTION
Lectin Gene construct and cloning:
The amino acid sequence derived from the MALDI MS/MS and x-ray crystallography data of the lectin purified from the soil fungus Sclerotium rolfsii formed the basis for the gene construct. The gene corresponding to this sequence SEQ ID 1 was chemically synthesized by following the standard protocols. The assembled gene was first inserted into a pUC57 vector. The insert was released by digesting the plasmid with Ndel & BamHI and then recloned in to pET20b previously digested Ndel and BamHI. After ligation the plasmid was used to transform E.coli DE3 (GOLD) host strain for expression. The recombinant clones were analyzed for release of insert after digestion with Nde I and Bam HI. The SDS-PAGE


analysis of recombinant E.coli upon induction with IPTG showed the expected ~16kDa protein.
Cell growth:
A single colony of the recombinant E.coli was inoculated into 5ml LB-Amp allowed to grow at 37°C overnight with shaking. The overnight grown culture was inoculated into the Fermentation Media and grown at 37° C till an OD of -2.0 was reached, the cultures were then induced with 250mM IPTG (final concentration) and grown overnight at 20°C.
Cell extract preparation:
The overnight grown cultures were centrifuged at 8000rpm for 10min and the pelleted cells (4gms) were suspended in 40ml of 50mM Tris-HCl pH8.0 containing ImM PMSF and ImM EDTA. The cell suspension was sonicated for 20min using the sonicator. The cells were centrifuged at 12000rpm, 10min at 4°C. The supernatant was used for further purification.
Purification of the protein:
DEAE-cellulose chromatography:
The supernatant (400mg of protein) in 50 mM Tris-HCl buffer pH 8.0 was loaded on 25ml DEAE (BIORAD) column equilibrated in 50mM Tris-HCl buffer pH 8.0. The column was washed with 2 column volumes of buffer followed by step-wise elution of the column with buffer b containing 75mM NaCl and 200mM NaCl. The lectin was eluted with buffer containing 300mM NaCl. This fraction contained 216 mg of protein.

PEI chromatography:
The DEAE elute (216mg) was loaded on to the NUCLEOSIL 4000-7-PEI (250 x 10mm) column, equilibrated with 50 mM Tris-acetate buffer pH 8.0. The bound protein was eluted by applying a linear gradient of NaCl from 0-100% in 15min at 2ml/min flow rate using the FPLC system and the protein eluted at 98%B (60mg). The eluted protein was dialyzed against water and repurified on the same PEI column using ammonium bicarbonate buffer.
The PEI column was equilibrated with 20mM ammonium bicarbonate buffer pH 8.0 and the protein was loaded on the column. The bound protein was eluted using buffer gradient from 20-500mM ammonium bicarbonate buffer. The eluted protein (30mg) was dialyzed against water, lyophilized and stored at -20°C.
Gel filtration chromatography: The final purification of the recombinant protein was achieved by gel filtration chromatography on Superdex G-75 equilibrated with 25 mM TBS, pH 7.2 on AKTA Prime plus purification system.
The expression of the protein and the purification of the protein were checked on 15% SDS PAGE and the results are given here. There was a major Coomassie blue stainable protein band corresponding to a molecular size of 16kDa.


EXAMPLES
Example 1: Identification of neoglycans expressed on activated and transformed lymphocytes in comparison with normal lymphocytes by the recombinant lectin;
In order to identify the neoglycoproteins expressed on lymphocytes upon activation by PHA and transformed cells, the interaction studies were carried out with respective cell membrane proteins with biotinylated lectin.
Isolation of Cell Membrane Proteins:
Human peripheral lymphocytes were prepared from the blood collected from a healthy donor. The blood sample (20ml) collected in to glass centrifuge tube containing EDTA (12.5 mM final concentration) and the normal lymphocytes were separated by centrifugation in Ficoll-Hypaque (Pharmacia) gradient. A portion of normal lymphocytes were activated by incubating with PHA (2.5 jig/ml PHA) in RPMI-1640 medium supplemented with 10% FCS for 72 hours in CO2 incubator. Activated cells were harvested by centrifugation and the activation was confirmed by checking for the expression of CD25 receptors.
Normal lymphocytes, activated lymphocytes and leukemic cell lines; Molt-4 and Jurkat were finally maintained in RPMI 1640 culture medium, supplemented with glutamine (2 mM), heat inactivated FCS (10%), penicillin (100U/ml) and streptomycin (100-ug/ml). The cultures were maintained at 37°C in humidified atmosphere (95% air and 5% C02). Membrane proteins from these cultured cells were isolated by using "Pierce


Membrane Protein Extraction Reagent kit" method (Mem-PER, Prod #89826) and the protein contents were estimated using Bio-Rad's DC-Protein estimation Kit. Isolated membrane proteins were finally concentrated by chloroform-methanol precipitation, and the precipitated proteins were air dried and stored at 4°C till further use.
Electrophoresis & western Blotting:
Polyacrylamide gel electrophoresis was performed in the presence of SDS using a minigel system (Hoefer Scientific Instruments, USA) in 10% gel for 60 mins at 120 volts. The proteins were transferred from polyacrylamide gels to Nitro Cellulose membranes in a semidry blotting equipment using transfer buffer (240mM Glycine, 25mM Tris, 0.1% SDS) containing 20% methanol at 75mA for 3hr at 4 °C. Membranes were saturated after transfer with blocking solution (3% p-BSA) and washed with TBST. Lectin binding was carried out at room temperature for 4 hours with biotin-coupled lectin at the final concentration of 20mg/ml (prepared in TBST). After thorough washing, blot was incubated with Streptavidin-HRP (1:1000 in TBST) at room temperature for 1 hr and excess Streptavidin-HRP was washed with TBST. Finally the lectin-glycoproteins bands were visualized by developing with DAB chromogenic system.
The results of these findings show that the present recombinant lectin has the ability to bind to some of the uniquely expressed neoglycans on the leukemic cells and also activated lymphocytes.


EXAMPLE 2: Identification of Lectin binding glycoproteins of the Ovarian Cyst fluid
Ovarian cyst fluid was collected aseptically from a patient admitted to SDM college of Medical Sciences and Hospital. The fluid was diluted thrice with PBS (50 mM, pH 7.2) containing ImM PMSF and centrifuged at 10,000 rpm, for 20 minutes at 4 °C to obtain the clear supernatant. Clear supernatant was filtered through a membrane filter (0.25 urn, Millipore Ind. Ltd.) and dialyzed first against PBS (50 mM, pH 7.2) and subsequently with several changes of deionized water. After extensive dialysis fluid was distributed in small vials and freeze dried and stored at -20° C till further use.
SDS PAGE and lectin blotting
Proteins from the ovarian cyst fluids samples were separated by SDS PAGE in 10 % gels as described by Laemmli et al using Hoefer Scientific Instruments, San Francisco, USA. Electrophoresis was carried out for 70 min.l using 110 volts. Immediately after the electrophoresis fractionated protein bands were resolved were blotted onto nitro cellulose membrane using semidry blot assembly. Blotted membrane was washed with distilled water and stained with Ponceau reagent to confirm the efficiency of blotting and to mark the location of standard proteins. Finally the blot was washed with TBS containing 0.1 % Tween -20 (TBST) and treated with 3 % P-BSA in TBST for overnight to prevent non-specific binding. After washing with TBST, blot was incubated with biotinylated lectins (20 mg/ ml in TBST) at 37° C for 4-6 hours. Excess and unbound lectin was removed by washing the membrane with TBST and then the membranes were incubated with


Streptavidin-HRP (lug/ml in TBST) for one hour. Unbound Streptavidin-HRP was removed by washing with TBST. The lectin binding glycoproteins were visualized by staining for peroxidase activity using DAB system.Red cell agglutination assay:;
The results of these investigations indicted the binding of the present lectin to several glycoproteins present in the ovarian cyst fluid.
Hemagglutination assay:
The hemagglutinating activity of the lectin was routinely assayed by serial two fold dilution technique of Liener and Hill ( J.Nutr., 49, 609-620, 1953) in microtuter plates using trypsinised human erythrocytes. The highest dilution of the lectin preparation causing visible hemagglutination was regarded as titer and the protein content in the highest dilution causing visible agglutination as 1 unit of hemagglutination activity. The specific activity is expressed as units per milligram of protein.


SEQ ID 1:
Ac-T-Y-K-I-T-V-R-V-Y-Q-T-N-P-D-A-F-F-H-P-V-E-K-T-V-W-K-Y-A-N-G-G-T-W-T-I-T-D-D-Q-H-V-L-T-M-G-G-S-G-T-S-G-T-L-R-F-H-A-D-N-G-E-S-F-T-A-T-F-G-V-H-N-Y-K-R-W-C-D-I-V-T-N-L-A-A-D-E-T-G-M-V-I-N-Q-Q-Y-Y-S-Q-K-N-R-E-E-A-R-E-R-Q-L-S-N- Y-Q-V-K-N-A-K-G-R- N-F-Q -I-V-Y-T-E-A-E-G-N-D-L-H-A-N-L-I-I-G-COOH
SEQ ID 2:
DNA sequence for lectin:
CAT ATG GTG TAT AAA ATT ACC GTT CGC GTG TAT CAG ACC AAT CCG GAT GCG TTT TTC CAT CCG GTG GAA AAA ACC GTG TGG AAA TAT GCG GAT GGC GGT ACC TGG AGC ATT ACC GAT GAT CAG CAC GTT CTG ACC ATG GGC GGT AGC GGC ACC AGC GGC ACC CTG CGC TTT CAT GCG GAT AAT GGT GAA AGC TTT ACC GCG ACC TTT GGC GTG CAT GAT TAT AAA CGT TGG TGC GAT ATT GTG ACC GAT CTG GCG GCG GAT GAA ACC GGT ATG GTG ATT AAT CAG GAA TAT TAT AGC GAA AAA GAT CGC GAA GAA GCG CGC GAA CGT CAG AAT AGC AAC TAT GAA GTG AAA GAT GCC AAA GGT CGT AAC TTT GAA ATT GTG TAT ACC GAA GCC GAA GGT AAT GAT CTG CAT GCG GAT CTG ATT ATT GGC TAA TAA GGA TCC
The length of the optimized sequence is 441 nucleotides which includes the 5' end Nde I (CATATG) and 3' end BamHI (GGATCC) restriction sites.

Protein Sequence: The number of amino acid coding for this lectin is 141 and is represented by SEQ ID 3.
SEQ ID 3:
VYKITVRVYQTNPDAFFHPVEKTVWKYADGGTWSITDDQHVLTMG GSGTSGTLRFHADNG 60
ESFTATFGVHDYKRWCDIVTDLAADETGMVINQEYYSEKRDEEARE RQNSNYEVKDAKGR 120
NFEIVYTEAEGNDLHADLIIG 141