Extraction and partial purification and sequencing of thaumatin-like ...

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Extraction and partial purification and sequencing of thaumatin-like ...

Extraction.......

Sindhuri Madineni.

family, have been found in the crop species barley (Hordeum vulgare L.) 8, rice (Oryza sativa) and wheat (Avena

sativa) 9 . They are named after their amino-acid sequence and structural similarities to the sweet tasting protein

thaumatin from the fruits of the West African rain forest shrub Thaumatococcus daniellii Benth 10 of the West African

rain forest. Based on their molecular weights, TLP’s can be divided into large and small types. These large type TLP’s

ranges from 21 to 26 KD with 16 conserved cysteine residues. The molecular mass of small group (present mainly in

conifers and cereals) is around 16 to 18 KD and has only 10 conserved cysteine residues because of a peptide deletion

11 . These cysteine residues forms disulfide bridges and are responsible for the heat and P H resistance 12 .

TLPs exert an antifungal effect through membrane permeabilization or a pore forming mechanism 13 . Occurrence of

positive charges on the TLPs surface enables them to interact with the surface of the yeast plasma membrane, while

hydrophobic interactions are responsible for the increase in permeability 7 . Later studies indicated that TLPs react with

the cell wall rather than with the membrane. Some TLPs bind to beta-1, 3-glucan and exhibit glucanase (laminarinase)

activity, but possessing glucanase activity does not necessarily mean antifungal activity 14 .

MATERIALS AND METHODS

Protein extraction and purification: Barley grains both soaked and unsoaked was grounded into fine paste and

powder in mortar and pestle. 1g. of each was homogenized with 5ml extraction buffer (0.2M Sodium acetate, 1.4

Sodium chloride and PVP, pH 5.5), and extract was collected by centrifugation at 6000 rpm for 20 min. The protein

extract was subjected to fractional precipitation with ammonium sulphate, this was then centrifuged at 6000 rpm for

10 min and pellet was collected. The pellet was dissolved in 20 mM Tris HCl. This extract was subjected to dialysis in

dialysis bag which were pre treated with sodium bicarbonate and boiled in water. Samples were loaded into these bags

and suspend them in water, without disturbing for overnight in refrigerator. These dialyzed samples were loaded onto

a DEAE-cellulose column equilibrated with NaCl buffer. Proteins were eluted with 0.2M to 1M NaCl dissolved in

20mM Tris HCl, pH5. The collected elutes were then subjected to protein estimation and molecular weight

determination.

Protein estimation: The protein concentrations were estimated using the Lowry’s method with BSA as standard. For

pure TLP samples, protein concentrations were determined colorimetrical OD at 650nm.

Purity test and molecular weight determination: Analytical SDS-PAGE was performed on a polyacrylamide gel

(4% (w/v) stacking gel and 10% (w/v) resolving gel), both in reducing and non-reducing conditions. Molecular weight

markers were: lactalbumin (14 kDa), trypsin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa)

and human sera albumin (67 kDa). After SDS-PAGE and IEF, proteins were stained with Coomassie Brilliant Blue G-

250.

Protein sequencing: The antifungal protein was identified by MALDI TOF. TOF ULTRAFLEX. Protein sequences

homology searches were applied using BLAST compared the protein sequence with known proteins in the database at

NCBI.

DNA Isolation: DNA was isolated using the buffer 0.1M Tris, 0.05M EDTA, 1.25% SDS. The DNA isolated was

quantified using spectrophotometer.

PCR amplification: All reactions took place in 200 µL PCR tubes containing 10 × PCR buffer (Bioserve

biotechnologies India Pvt. Ltd, Hyderabad), 2.5 mmol L−1deoxynucleotide triphosphates(dNTPs), 25 mmol

L−1MgCl2, 10 pmol Lprimers, 1.0 U Taq DNA polymerase (Bioserve biotechnologies, Hyderabad) and 1 µL DNAcontaining

solution obtained using the extraction procedure described above. PCR was performed on an Eppendorf

Personal Mastercycler (EppendorfAG, Hamburg, Germany) with the following program: a 3 min step at 94 o C and 35

cycles of 30 sec at 94 o C, 30 s at 40 o C annealing temperature, and 60 sec at72 o C, followed by a final extension step at

72 o C for10 min. The primers were designed using primer3 software by comparing the sequences retrieved from NCBI.

The forward and reverse primers were 5’-GCGTACAGTTACCCCAAGGA-3’ and 5’-

TGTATGCATCCAAACGCACT-3’ respectively.

DNA sequencing: DNA was sequenced with MEGA BASE 1000 automated sequencer using the ABIPRISM Dye

Terminator Cycle Sequencing Ready reaction kit (PerkinElmer, Foster City, CA, USA) following the manufacturer’s

specifications. DNA sequences and amino acid sequences were compared with sequences present in the GenBank and

EMBL databases using the BLAST mail server.

Anti fungal and bacterial activity: Antifungal and bacterial activity was performed by agar well diffusion method on

NAM and PDAM plates against fungi like Candida albicans and bacteria like Bacillus subtilis, Saccharomyces

cerevisiae, E. coli. Nutrient agar media plates were prepared and 100µl of culture was spreaded on each plate with

274 J. Chem. Bio. Phy. Sci. Sec. B, Nov. 2011- Jan. 2012, Vol.2, No.1, 273-278

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