THEREOF

FIELD OF INVENTION

The invention relates to the field of carbohydrate binding proteins, and more specifically to a protein which binds specifically to particular oligosaccharides associated with particular medical significance. The invention specifically relates to lectins, more particularly to recombinant fungal lectins and method of preparation thereof.

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. . Because of their selective cell specificity. lectins may serve as carriers for targeted drug delivery. Some of the lectins also show anti tumor activity. 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 Galcrt , 3Gal and Galcd , 3Galp1 , 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)

Peanut lectin that shows affinity to Thomsen-Friedenreich antigen has 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 1 1.73 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).

Lectins thus have enormous potential in the area of medical assays. The instant application proceeds to provide certain lectins with novel characteristics so as to be useful in the medical field.

SUMMARY OF THE INVENTION The present invention provides a method for preparing a recombinant lectin expressed in a host cell such as E.coli or yeast. This method comprises the synthesis of a lectin gene based on amino acid sequence derived from the MALDI MS/MS of the Sclerotium rolfsii lectin (SEQ ID.1), a soil borne phytopathogenic fungus, and its cloning in the host cell. 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 A1 cell and has sugar specificity towards GalNaca1 -3 (Fuca1-2) Gai -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. The amino acid sequence SEQ ID 3 represents such lectin having all the above characteristics. The nucleic acid molecule encoding the amino acid for the protein of the above characteristics is also within the scope of the application. The DNA sequence of SEQ ID 2 represents such nucleic acid encoding the lectin having the above characteristics. The sequences also envisage any change in the nucleotide or amino-acid sequences including but not limited to any substitution, deletion, addition or modification such as acetylation, nitration, pegylation,sulphonation etc. in any of the positions of the entire sequence and also to include truncations at 5' or 3' end of the sequences which do not alter the properties disclosed in the instant application.

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. The growth modulatory effect of the recombinant lectin can have potential application in cancer therapy

In another aspect the recombinant lectin can be used as a drug delivery agent for cancer treatment. The application particulary discloses a DNA sequence (SEQ ID 2) 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.

The method for producing said recombinant lectin has also been disclosed. The cloned lectin is expressed after induction with the inducer. IPTG. The expressed protein appears in soluble and active form. The recombinant lectin is 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 calculated PI value is 4.89.

The recombinant lectin exhibits specificity towards blood group A1 and can be used for blood typing. The lectin binds specifically to human breast, colon and ovarian cancer tissues and is 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 for affinity purification of 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.

BRIEF DESCRIPTION OF FIGURES

Figure 1 : The Amino acid sequence of lectin protein isolated from S. rolfsii (SEQ ID 1) Figure 2: A DNA sequence encoding the lectin of the instant application, SEQ ID 2. The length of the optimized sequence is 441 nucleotides which includes the 5" end Nde I (CATATG) and 3' end BamHl (GGATCC) restriction sites.

Figure 3: Protein Sequence of the recombinant lectin of the instant application. The number of amino acid coding for this lectin is 141 and is represented by SEQ ID 3. The altered/modified positions have been highlighted in the sequence.

Figure 4: Multiple sequence alignment (CLUSTAL 2.0.3) of the SEQ ID 1 and SEQ ID 3.

DETAILED DESCRIPTION OF THE INVENTION

The instant application provides for altered/modified lectin protein of fungal origin. The lectin purified from the soil fungus Sclerotium rolfsii is the basis of the invention. An altered form of the gene sequence of the native S.rolfisi has been cloned and subsequently expressed as a recombinant protein. The complete description in terms of experimental data is herewith provided for one representative host E. coli:

The words modify and alter have been interchangeably used in the specification to indicate any change in the nucleotide or amino-acid sequences including but not limited to any substitution, deletion, the modification to include truncations at 5' or 3' end of the sequences which may result in a protein of reduced size but retaining the bioactivity. The word modification would also include chemical modification of the derived recombinant protein by resorting to pegylation, nitration, glycosylation, phosphorylation etc.

Lectin Gene construct and cloning:

The amino acid sequence derived from the ALDI 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 250μΜ 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-HCI pH8.0 containing 1 mM PMSF and 1 mM 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. At higher growth temperatures substantial amount of the recombinant protein was found in the inclusion body fraction.

Purification of the protein:

DEAE-cellulose chromatography:

The supernatant (400mg of protein) in 50 mM Tris-HCI buffer pH 8.0 was loaded on 25ml DEAE (BIORAD) column equilibrated in 50mM Tris-HCI 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 NaCI and 200mM NaCI. The lectin was eluted with buffer containing 300mM NaCI. 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 NaCI 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 another PEI column preequilibrated with 20mM ammonium bicarbonate buffer pH 8.0.

The bound protein was eluted using buffer gradient from 20-500mM ammonium bicarbonate pH 8.0. 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 expressed protein and the purified fractions were analyzed on 15% SDS PAGE. The purified fraction was visualized by staining with Coomassie blue. The purified protein appeared to be essentially pure and corresponded to a molecular size of about 16.kDa.

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 pg/ml PHA) in RPMI-1640 medium supplemented with 10% FCS for 72 hours in C0 ₂ 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% CO ₂ ). 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 20pg/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 1 mM 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 pm, 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.

SOS 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. using 1 10 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 pg/ 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 (1 pg/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. Results of the hemagglutination assay show that the recombinant lectin agglutinates only the red blood cells of A1 blood group human individuals, not those of B or O group. Also the activity of agglutination by the recombinant lectin is two and half times higher than the Dolichos biflorus lectin

Recombinant Sequences

The gene sequences and the proteins having the following biological activity: specific binding to blood group A1 cells, sugar specificity towards GalNaccrt-3 (Fuccrt-2) Gaipi-R and N-acetyl galactosamine, Tn antigen, no specificity towards TF antigen, substrate specificity towards N-acetyl galactosamine, mucin and feutin, and specific binding to breast cancer colon cancer, ovarian cancer and leukemic cells are covered within the scope of the application. The nucleotide molecules encoding the recombinant molecules may be DNA or RNA. The proteins of the above mentioned characteristics whether obtained by deliberate or random mutations are covered within the concept of the instant application.

In one of the preferred embodiments, the expressed lectin protein (SEQ ID 3) differs from the native lectin protein (SEQ ID 2) in terms of atleast one amino acid modification/substitution at positions shown in figure 4. Of these, positions 1 , 34, 109 have amino acid substitutions: T to V, T to S and L to N respectively. There are other modifications such as Asparagine to Aspartic acid and Glutamine to Glutamic acid substitutions.

The recombinant lectin sequences can be biotinylated or attached with fluorogenic chromophore by using commonly known methods to be used for the detection of cancer cells or cancer associated specific antigens. The growth modulatory effect of the recombinant lectin finds its application in cancer therapy. Further the use of recombinant lectin as a drug delivery agent for cancer treatment is envisaged in the present application.

While the present invention has been described with respect to certain preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the following claims.