Azurin yield from different strains (
The use of microorganisms and their products as possible therapeutic agents in the control of cancer begins at the latter part of the nineteenth century. The search of new drugs from microbial sources against infectious disease has been augmented when Alexander Fleming (1928) discovered penicillin . The secondary metabolites from microorganisms play a vital role in developing antibiotics and chemotherapeutics [2, 3]. Several researchers have reported various anticancer molecules from different microbial sources . Even though chemotherapy is efficient in enhancing patient survival with primary tumors continue to have deprived prognosis. The rapid advances in the field of antibiotics have inspired new hope that the search among biological systems will disclose a chemical agent which will exert a destructive effect upon neoplastic growth without seriously affecting normal cells. Using live or attenuated pathogenic bacteria or its metabolites in treatment of cancer excretes toxic effects among patients. Azurin, a redox protein recently fascinated biomedical researcher’s immense interest as an anti cancer therapeutic agent which enters human breast cancer cells and induces apoptosis without any adverse effects in cancer patients . Azurin, a secondary metabolite derived from bacterial species especially from
Previous researchers  adopted genetic engineering techniques and other bacterial species for purification of azurin. This study is concerned of enhanced azurin synthesis from different strains of
2. Materials and methods
2.1. Chemicals and reagents
Growth medium constituents were of analytical grade obtained from Hi-Media laboratories, India. The buffer ingredients were purchased from Merck Chemicals Ltd, India. Sephadex G-25, G-75, diethyl amino ethyl cellulose (DEAE) cellulose and carboxy methyl (CM) cellulose were all obtained from Sigma-Aldrich, USA. The 3, 5-dimethoxy, 4-hydroxy cinnamic acid otherwise called as sinnapinic acid a matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) was also acquired from Sigma-Aldrich. Protein concentrations were measured by Lowry’s method with bovine serum albumin as standard. Standard dialysis bag with 3 kDa cutoff was purchased from Sigma-Aldrich. Powder of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), propidium iodide (PI) and dimethylsulfoxide (DMSO) solution were procured from Sigma Aldrich, India. Cell culture media and other constituents of media are purchased from Hi-media Laboratories Ltd, India. Fetal bovine serum was obtained from Invitrogen Life Technologies, USA.
2.2. Cultivation of P. aeruginosa MTCC 2453
A freeze dried culture of
2.3. Impact of copper sulphate and potassium nitrate on culture medium
2.4. Extraction of cellular protein
After 21 hrs incubation, cells were harvested by centrifugation method at 13200 g for 15-20 minutes by using ultra centrifuge (Eppendorf, Hamburg, Germany). Cell pellets was collected and suspended in the appropriate volume of 0.02M potassium phosphate buffer pH 7 with protease inhibitor and kept in the ice basket for sonication. Cells were sheared by Ultra sonicator (Cole Parmer, USA) of approximately 100 ml batches of cell suspension. All batches were sonicated for 1-2 minutes at 100W. After sonication the samples was stirred vigorously and centrifuged at 10000g for 20 minutes which removes cell wall debris. The green-brown crude supernatant was stored. Resuspended the precipitate in same buffer, stirred it vigorously and centrifuged as before and the supernatant were stored with the previous extracts [11, 12].
2.5. Ammonium sulfate precipitation of proteins
The Crude (supernatant) was saturated to 45% (277g/l) by slowly adding ammonium sulfate salt at 40C for precipitation, kept it for overnight [5,6]. After precipitation the solution was centrifuged at 20,000g for 25 minutes [6,]. Collected the yellow supernatant saturated again to 95% by adding (NH4)2SO4 (372g/l) slowly and kept at 40C for overnight. The overnight precipitated solution was centrifuged at 23000g for 45 minutes. Pale supernatant was discarded. Precipitate (contains azurin) were collected and resuspended in 0.02M Potassium Phosphate buffer pH 7[11, 12].
2.6. Dialysis of the supernatant
Azurin suspended in 0.02M potassium phosphate buffer pH 7 was dialysed by standard dialyses bag purchased from Sigma-Aldrich, (Kolkata, India) having 3 kDa molecular weight cut off at 40C for 20 hours on the same buffer for overnight with continuous gentle stirring. Dialysis was done until the solution attains its buffer pH. The solutions were kept at 40C after dialysis for further purification [11, 12].
2.6.1. Purification of Azurin on Ion – Exchange chromatography
Dialysate (contains azurin) were initially treated with DEAE. 100 ml slurry of DEAE cellulose equilibrated in 0.02M potassium phosphate buffer pH 7 were treated with the dialysate and stirred for 20-30 min at 40C.The suspension was centrifuged at 10,000g for 15 min. Azurin does not adsorb in the gel remains in supernatant but most of the unwanted proteins like yellow flavo proteins are removed. Supernatant was collected. DEAE cellulose precipitate was resuspended in the same buffer and again centrifuged at 10,000g for 15 minutes to remove all unattached proteins [11-13].
The supernatant after DEAE treatment was saturated to 100% (766g/l) with (NH4)2SO4 at 40C for overnight for precipitation. After saturation, precipitates are mixed gently and kept for centrifugation at 10000g for 10 min. supernatant was collected for dialysis at 40C for overnight with gentle stirring with the same before. Dialysis was continued till the solution pH attains its buffer pH [11-13].
2.6.2. Purification of Azurin on gel-filtration chromatography
Sephadex G-25 beads were equilibrated in the 0.02M potassium phosphate buffer pH 7 [Parr S R et al 1976] for overnight, and tightly packed in 3cm x 25cm length glass column without any bubbles. The column was initially washed with 0.02M potassium phosphate buffer for twenty volumes of the gel packed. The Flow rate was adjusted to one minute per ml. slowly the dialysate (after DEAE treatment) was added with the eluent buffer 0.02M potassium phosphate buffer pH 7 on the column. Thirty fractions were collected at one minute interval [12-14].
Sephadex G-75 beads in powder form are equilibrated in 0.01M Tris/Hcl buffer pH 7.5 for overnight. After equilibration the beads were tightly packed in a 3cm x 45cm glass column. The column was washed with same equilibrating buffer for fifty volumes of the column value. After washing with buffer one ml of the sample (fraction (a) collected from the G-25) were passaged and eluted with the same equilibrating buffer. Seventy five fractions were collected at 1 ml/6minutes flow rate [12-14].
2.6.3. Purification of Azurin on ion – Exchange (anionic) chromatography
The CM cellulose beads from Sigma–Aldrich (Kolkata, India) were equilibrated for overnight in the ammonium acetate buffer pH 3.9 adjusting the pH by 0.05M acetic acid with 2M NH3.After swelling, the beads were packed in a 5cm x 15cm glass column and washed for ten times of the column volume. Gently one ml of the sample added (Fraction (e) collected from G-75) over the top of the column and left it for 5-10 minutes to bind the protein inside the beads. After 10 minutes the column was eluted with ammonium acetate buffer pH 4.65 [12-14].
2.6.4. Characterization of Azurin (purified from P. aeruginosa MTCC2453)
The most successful method to analyze biopolymers such as, proteins, peptides, sugars and large organic molecules which are tend to be fragile and fragment when ionized by more conventional ionization methods . The Fractions collected from G-25, G-75 and CM cellulose were performed MALDI for molecular weight determination. Two micro liter from each fraction of the chromatography was added with 20µl of 3, 5-Dimethoxy, 4-Hydroxy cinnamic acid otherwise called as sinnapinic acid (Sigma-Aldrich. Kolkata, India). Tiny spots were made on silver plate and kept for drying for 4-6 hours to drain the water molecules. Further spots were dried with a vacuum drier to make a crystalline molecule. After drying the samples were placed in the MALDI-ToF chamber (Voyager De pro, applied systems Illinois, USA) for analysis by using nitrogen laser at 337 nm.
2.6.5. Purification profile of Azurin ((synthesized from P. aeruginosa MTCC2453) by SDS-PAGE
Five ml of 12% resolving gel contains 1ml distilled water, 30% acryl amide, 1.5M Tris (pH 8.8), 10% SDS, 10% APS and 0.002µl TEMED for polymerization was casted in the glass slab without any bubbles and kept it for 10-15 minutes. After polymerization of the resolving gel, 3ml of stacking gel (4%) were loaded over the resolving gel which contains 0.68 ml distilled water, 30% acryl amide, 1M Tris (pH 6.8) 10% SDS,10% APS, and 0.001ml TEMED. After casting the gel, proteins purified from different chromatography were loaded with bromophenol (molecular weight marker dye) at different lanes for profiling the protein purification process.
Glass slab gel were kept in the electrophoresis tank with tank buffer (196 mM glycine, 0.1%SDS, 50mM Tris-Hcl pH 8.3 made by diluting a 10x stock solution). This setup was connected with power pack initially in 80mV to 100 mV. After running the gel up to its anode end, was removed and stained with 0.2% coomassie brilliant blue for overnight. Destained with destaining solution (45: 45: 10 – methanol: water: acetic acid) which destains the comassie blue until it reveals the bands. The bands (figure 4) were observed under UV transilluminator (Biorad, PA, USA) .
2.6.6. FTIR analysis
Infrared spectroscopy experiments were performed using a Nexus 870 (Thermo Nicolet Corporation, Madison, USA) spectrometer equipped with a potassium bromide (KBr) beam splitter and DTGS (deuterated triglycine sulfate) detector in the range of 3,000-4000 cm−1. We recorded 32 scans per spectrum at a 2 cm−1 resolution for 100 µl of azurin liquid samples in 0.02 M PBS buffer (pH 7.0). We kept the same buffer as a background medium and performed all measurements at room temperature. We corrected spectra for the moisture and carbon dioxide in the optical path. The curves were deconvoulted and imported into Omnic’s peak fit software (Thermo scientific, Illinois, USA) and a Gaussian curve fitting performed .
3.1. Growth of P. aeruginosa strains
The inoculated growth of
3.2. Effect of copper sulphate and Nitrate in azurin synthesis
Earlier studies showed that azurin production by different bacterial strains were similar to the azurin produced by
|Total dry cell yield in g/l medium||1650||1780||1590|
|Protein concentration after 45/95 % (NH4)2SO4 precipitation (g/l)||1520/1410||1460/1550||1205/1300|
|Protein concentration after DEAE treatment in g/l medium||560||610||490|
|Protein concentration after G-25 treatment g/l.||440||485||384|
|Protein concentration after G-75 treatment g/l.||320||350||302|
|Total Azurin synthesis in mg/gdry bacteria. (CM cellulose)||2.9||2.4||2.6|
3.3. Chromatography methods for azurin Purification
DEAE and G-25 are gel filtration columns which remove positively and negatively charged proteins respectively. The unwanted flavo proteins and positively charged proteins were removed during DEAE chromatography. The collected fractions from G-25 were quantified for protein concentration in the UV-Spectrophotometer at 280nm wavelength. Azurin and other proteins more than 5 kDa were eluted immediately after void volume is plotted as graph (Figure 3.).
Peak (a) from G-25 was loaded on G-75 for further purification. The G-75 fractions were quantified for protein concentration in the UV-Spectrophotometer at 280nm wavelength. Azurin a 14 kDa protein will elute after binding in to the beads when the elution buffer elutes it. Thus, azurin and some other proteins will elute very lately, was confirmed from the OD values of the spectrometer, when plotted as graph (Figure 4). The azurin will form a thick band when passages through CM cellulose column which was eluted by ammonium acetate buffer pH 4.65. Ten fractions were collected and absorbed under UV spectrometer at 280nm wavelengths for azurin concentration (Figure 5.).
3.4. Characterization of Azurin (Purified from P. aeruginosa MTCC2453)
In this study we profiled our purification process at every step by MALDI-ToF (Figure 6.) and SDS-PAGE (Figure 7.) and to confirm the azurin presence in our experiments. Cellular proteins loaded in lane 2 of SDS-PAGE reveals whole cell proteins of
3.5. FTIR analysis
The functional groups of azurin were studied using FTIR spectrum. The presence of the amide I band was indicated by the peak around 1650 cm-1 region, which arises primarily because of the stretching vibration of the main chain of carbonyl groups in the protein backbone coupled with the in-plane N-H bending and C-N stretching modes. Furthermore, the presence of an amide band around 1650 cm-1 signifies α-helix secondary structure of azurin. Azurin synthesized from all strains showed a significant shift in the amide I band with one another, indicating differences in their helix secondary structure of azurin. The most prominent among all strains is
Azurin production from
The fraction collected from G-25 containing only more than 5 kDa proteins were passed through on G-75 which has 5-80 kDa fractionation range. The higher proteins above 80 kDa molecular weight elute after void volume; the remaining proteins between 6-70 kDa were bounded within the beads later eluted by the buffer. The fraction which showed 14 kDa molecular weight by analyzing in MALDI-ToF spectrometer for all the fractions (MALDI-ToF results not shown for all fractions which showed peak) were collected and again purified in CM cellulose chromatography. The fraction which showed peak in CM cellulose was again observed in MALDI-ToF spectrometer to confirm the presence of 14 kDa molecular weight of Azurin.
Our idea of adding copper in the culture medium was not only for the enhanced azurin synthesis, but to reveal the differences of azurin’s stability in the secondary structure for all
MALDI-Matrix-Assisted Laser Desorption/Ionization, SDS-PAGE-Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, FTIR- Fourier Transform Infrared Spectroscopy, CuSO4 – copper sulphate, KNO3 – Potassium nitrate, MTT -3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, PI-Propidium Iodide, DMSO-dimethylsulfoxide, MALDI-ToF-Matrix-Assisted Laser Desorption/Ionization-Time of Flight, MTCC- Microbial Type Culture Collection center, CM-carboxymethyl, DEAE-Diethylaminoethyl Cellulose