Journal of Pharmacy Research Vol.4.Issue 5. May 2011 V.Shanmugaraju et al. / Journal of Pharmacy Research 2011,4(5),1533-1534 1533-1534 Research Article ISSN: 0974-6943 Available online through http://jprsolutions.info * Corresponding author. V.Shanmugaraju Department of Biotechnology , DR NGP Arts and Science College , Coimbatore 48 ,India Isolation and partial characterization of anti cancer protein from Bacillus pumilus against HepG2 cancer cell line V.Shanmugaraju 1 *, N.Abirami 2 , A.Mahalakshmipriya 1 and R.Revathy 1 1. Department of Biotechnology , DR NGP Arts and Science College ,Coimbatore 48 ,India 2. Department of Biochemistry, PSG Arts and Science College, Coimbatore 14 ,India Received on: 02-02-2011; Revised on: 08-03-2011; Accepted on:12-04-2011 ABSTRACT Bacillus pumilus was isolated from soil sample and confirmed by 16s rDNA sequencing. The anti cancer protein was obtained by ammonium sulphate precipitation and purified by Sephadox G100 column chromatography. The lyophilized fractions were screened for anti MRSA (Methicillin Resistant Staphylococcus aureus) and anti ancer activity against liver cancer cells (Hep G2) 150μg/ml of protein exhibited the high level of cyotoxicity (75%) towards the Hep G2 cells where the positive control cyclophospamide showed cytotoxicity of 76%. The anti cancer protein was characterized by 2D gel electrophoresis and peptide Mass Fingerprinting using MALTI TOF MS. Key words: Anticancer peptide, Anticancer protein, Antimicrobial peptides. INTRODUCTION The development of new classes of anticancer drugs that lack the toxicity of conventional chemotherapeutic agents and are unaffected by common mechanism of chemo-resistance would be a major advance in cancer treatment. Numerous chemotherapeutic drugs have been developed to treat cancers, including DNA alkylating agents, antimetabolites and hormone agonists/ antagonists. Although these drugs have been successfully used for the treatment of metastatic cancers, severe side effects and dose limitations are prevalent. As a result of their inability to distinguish between cancer cells and proliferating normal cells, current drugs kill both. More over cancer cells develop resistance to these drugs that is mediated by the over expression of the multidrug resistance proteins that pump the drug out of the cells and thus render the drugs ineffective [8]. Thus there is an urgent need to develop new classes of anticancer drug with new mode of action that selectively target the cancer cells.Cationic peptides are widely distributed in living organisms playing a variety of functions. They are often referred to as antibacterial or antimicrobial peptides due to their well characterized role in innate immunity against infectious agents [2]. While most of the studies on cationic peptides have focused on their antimicrobial activity, other biological effects are emerging and in the past few years the ability of some peptides to affect the tumor cells has been reported. [1,4,7,10].Most of these peptides have an amphipathic structure and they preferentially bind and insert into negatively charged cell membranes, consequently destabilization of the membrane disturbs the electrolyte balance and induces the intracellular contents to leak leading to cell death. In contrast to normal eukaryotic cells, which generally have low membrane potentials and whose outer leaflet almost exclusively consist of Zwitterionic phospholipids, the prokaryotic and cancer cell membranes maintain large transmembrane potentials and have a higher content of anionic phospholipids on their outer leaflet. Many AMP therefore preferentially disrupt prokaryotic and cancer cell membranes rather than eukaryotic membrane [6].The present study was undertaken to characterize and evaluate the anti cancer potential of peptide isolated from Bacillus pumilus a new classes of anti cancer peptide. MATERIALS AND METHODS Sample collection: The soil sample was collected from Kollam sea coast, Kerala, India. The samples were collected in sterile polythene bags and were brought to laboratory by preventing any contamination on the way. The sample was then stored at 4 0 C until used. Isolation of Bacteria: The soil sample was serially diluted by ten fold dilution. After serial dilution from each dilution 0.1ml of sample was taken and spread plated on Nutrient Agar. The inoculated plates were incubated at 34 0 C for 24hours. After the incubation period individual colonies were picked upon the basis of their macroscopic characteristics such as size, shape, surface, appearance, texture and color. And further the isolates were purified by repeated streaking and were sub-cultured on Nutrient Agar slants. Screening For Antibacterial Activity of Isolates by Point Inoculation Method: MRSA was obtained from Clinical laboratory, Kovai Medical Centre and Hospital, Coimbatore, TamilNadu, India. The over night grown cultures of MRSA in Nutrient Broth was uniformly swabbed on the surface of the Muller Hinton Agar plates using sterile cotton swabs. Then the isolates obtained from the soil samples were spotted on to the Muller Hinton Agar plates which are seeded with MRSA. The plates were incubated at 34 0 C for 24hours. After the incubation period the plates was observed for the zone of inhibition. The bacterium which showed the antibacterial activity was identified by 16S r DNA sequencing. Production and Purification of Antimicrobial Protein: For the production of antimicrobial protein the bacterium Bacillus pumilus was grown in 100ml Nutrient Broth medium at 37 0 C in a rotary shaker at 150 cycles/ minute. After cultivation for 48hours the cells were harvested by centrifugation at 6000 rpm for 15min- utes. The supernatant was sterilized with 0.45μm filter membranes and stored at 4 0 C. This was considered as cell free supernatant (CFS). The CFS was precipitated with ammonium sulfate at 30% (w/v) saturation (17.6 g/ 100 ml). The solution was incubated for 12hours at 4 0 C and protein was precipitated by centrifugation for 30minutes at 15,000 rpm. The precipitate containing protein was re suspended in 10mm Sodium Phosphate Buffer (pH 7.0) and desalted by using desalting column purchased from Genei Pvt. Ltd, Bangalore. The desalted protein solution was further purified by gel filtration chromatog- raphy on Sephadex G-100 column (1.4cm × 45cm) and eluted at a flow rate of 1ml min- 1. Fractions were also monitored for A280nm using spectrophotometer. The determina- tion of soluble protein was derived out by the Folin Phenol reagent method (Lowry et al, 1951) with Bovine serum albumin as standard. Fractions were lyophilized and tested for protein content and antimicrobial activity against the MRSA. The fractions which showed the antimicrobial activity against the MRSA was used for further characterization and to screen the anti-cancerous activity. MTT Assay: The HepG2 Cancer cells were grown in a 96-well plate in Delbucco’s minimum essential medium (DMEM, Hi Media) supplemented with 10% fetal Bovine serum (Gibco Labora- tories) and antibiotics (Streptomycin, Pencillin-G, Kanamycin, Amphotericin B). About 1mL cell suspension (105 cells/ mL) was seeded in each well and incubated at 37 0 C for 48 hours in 5% CO 2 for the formation of confluent monolayer. The monolayer of cells in the plate was exposed to various dilutions of protein obtained from B.pumilis. The cell viability was measured using MTT assay with MTT solution (5 mg/ mL) and DMSO. This tetrazolium salt is metabolically reduced by viable cells to yield a blue insoluble Formosan product measured at 570nm spectro photometrically [3]. Controls were main- tained throughout the experiment (untreated wells as cell control). The assay was per- formed in triplicate for each of the dilution. The mean of the cell viability values was compared to the control to determine the effect of the protien on cells and percentage of cell viability was plotted against concentration of each of the protein dilution The minimum concentration of the protein that was toxic to liver cancer cell was recorded as the effective drug concentration with compare to positive control (PC- Cyclophospamide). 2D Gel Electrophoresis Separation of Anti-Cancer Protein: 1mg of protein was loaded onto an IPG strip (7cm, 4-7 linear) by over night passive re hydration. The isoelectric focusing was carried out on a Bio-Rad Protean IEF cell using a programme (10hours at 50V, 15min at 250V, and 5hours at 8000V). Strips equilibrated twice for 10minutes each in 8M urea, 2% SDS, 0.05M Tris-HCl, pH 8.8, 20% glycerol. The first incubation contained 2% DTT and the second contained 2.5% Iodoacetamide. The strips were then layered on 12% Tris-Glycine –SDS gels and embedded in place with 0.5% Agarose. Electrophoresis was performed at a constant voltage of 200 V until the dye front ran off the gel. Gel was stained with Sypro Ruby stain.