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

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

Nov.2011-Jan.2012. Vol.2.No.1, 273-278 E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences

Research Article

An International Peer Review E-3 Journal of Sciences

Available online at www.jcbsc.org

Section B: Biological Sciences

Extraction and partial purification and sequencing of

thaumatin-like protein (TLP) from barley and it’s screening

for antimicrobial properties

Sindhuri Madineni

DVR & Dr.H.S. MIC College of Technology, Kanchikacherla, A.P, INDIA

Received: 18 November 2011; Revised: 26 November 2011; Accepted: 30 November 2011

ABSTRACT

Thaumatin like proteins (TLP’s) are the products of a large, highly complex gene family involved in host defense

mechanism and belongs to pathogenesis related proteins of group 5. TLP’s show homology with thaumatin, a

sweet tasting protein from fruits of Thaumatococcus danielli. TLP’s have glucan binding and glucanase activities.

The current study reported the extraction, purification and sequencing of TLP’s from Hordeum vulgare grains. A

protein with an apparent molecular mass of 22 KD, which was one of the most abundant proteins in extracts of

barley grains was purified by ion exchange chromatography and identified by amino acid sequence to be a

thaumatin-like protein. The partial sequencing of protein contains 111 aminoacids. The TLP coding gene was

sequenced by isolating DNA and amplifying with the specific TLP primers. The 181bp PCR amplified product was

sequenced. The comparison of sequence with the other TLP proteins gave 100% similarity. The antimicrobial

activity of TLP protein was tested against Candida albicans, Bacillus subtilis, E.coli, Saccharomyces cerevisiae as

they are pathogenic organisms. The results showed that it has maximum inhibitory affect against Candida

albicans.

Keywords: Thaumatin-like protein, Pathogenesis-Related protein, Barley, Antimicrobial activity.

INTRODUCTION

Plant proteins, induced in a pathological or related situation, named pathogenesis-related (PR) proteins, have been

classified into 17 families based on amino acid sequence, serological relationship and enzymatic or biological activity.

Although PR proteins were considered as inducible proteins elicited by pathogen attack and other stressors, they are

present constitutively in different plant organs including grains 1-,3. Pathogenesis related proteins were discovered in

1970’s by two independent groups 2 . They were detected in tobacco leaves infested with Tobacco Mosaic Virus. PR

proteins are usually defined as host specific proteins that are induced in several plant species during pathogen attack or

certain abiotic stress condition 3 and chemical exposure or by adverse environmental factors 4 . PR’s are low molecular

weight proteins i.e. 10-40 KDa which can survive in harsh environments due to their biochemical properties. They

remain soluble and very stable at low pH; they are relatively resistant against proteolytic cleavage. PR’s are

predominantly localized in the vacuole, cell wall and the intracellular space and they usually have extreme isoelectric

points 5 .

Originally, the PR’s were grouped into five families based on sequence characteristics. Now, this group of proteins

has been extended to 17 families 6, 7. Multiple isoforms of Thaumatin-Like Protein (TLP), which belong to the PR-5

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


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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

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Sindhuri Madineni.

different organism. Agar wells were prepared after 10 minutes and 100 µl of protein extracts were added in each well.

The plates were incubated at respective growth temperatures for 24- 48 hrs.

Results and Discussion: Multiple isoforms of Thaumatin-Like Protein (TLP), which belong to the PR-5 family, have

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

vinifera) were studied by various 1,13,15,16.

From the soaked and unsoaked barley seeds protein was extracted by using the sodium acetate buffer. Isolated protein

was further purified by using conventional methods such as ammonium salt precipitation, ion exchange

chromatography. For the protein purification by chromatography the buffer used was Tris Hcl with Nacl. The protein

molecular weight was determined using SDS-PAGE run with a standard marker and found to be approximately 22KD

as shown in the Fig.1. The molecular mass obtained corresponds to other TLP proteins reported by 1, 17 . The protein

isolated was assessed by colorimeter estimation using Folin-Ciocalteau with BSA as standard. The values of the

standards and TLP elutes obtained from ion exchange were measured at 650nm and shown in graphs with standard

and test values Fig.2.

The protein sequence obtained was found to be containing 111 amino acids as shown in Fig. 3. The protein sequence

obtained was compared with the TLP protein sequence retrieved from NCBI. The aligned result shows 95% similarity.

The result corresponds to the earlier work done by 8 .

Fig. 1: SDS PAGE

The band was found at 22KD in the gel when compared with the marker in first lane.

Fig.2: Graph for protein concentration

The graph was plotted for BSA standard values and soaked and unsoaked barley grains with concentration on X-axis

and optical density on Y-axis. The graph represents the highest concentration in soaked grain than unsoaked.

RTVTPRTTPPASSLAPAAQTTRSSSAHERPCMEEVSSTRTRISLCLSDVIYQNKCVWMHT

YITSDRHRLILVRVLETSSMHGRSWAEDDLVVCAAGASEDAGGVVLGVTVR

Fig. 3: Protein sequence

The TLP protein coding gene was amplified by using specific primers 5’-GCGTACAGTTACCCCAAGGA-3’ and

5’-TGTATGCATCCAAACGCACT-3’ designed by comparing TLP gene sequences from the other species. The TLP

protein coding gene sequences were retrieved from NCBI and compared using BLASTn. The similar sequences were

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Sindhuri Madineni.

aligned and forward reverse primers were designed using Primer3 software. These were used to amplify the TLP gene

of DNA isolated from barley seeds. The buffer used for DNA isolation yields a good amount and run on agarose gel

(as shown in Fig. 4) was quantified using spectrophotometer. The average DNA yield is approximately 15µg per

100mg of sample. The DNA purity was judged by the A260/A280 ratio which is 1.92. Thus the DNA isolation method

described yields a good quality of DNA without any protein contaminations.

Fig.4: DNA bands on Agarose gel

Fig. 5: PCR amplification

The DNA was amplified using TLP specific primers and the expected size of 181 bp was obtained as shown in fig 5.

The result corresponds to the earlier work done by 8 . The amplified product was sequenced (Fig. 6) on MEGA BACE

model 1000 and the sequence shows 100 % similarity with the TLP protein gene sequence. The blast analysis of gene

sequence was shown in Fig. 7.

GCGTACAGTTACCCCAAGGACGACGCCACCAGCATCTTCACTTGCCCCGGCGGCACAAACTACCAGGTC

ATCTTCTGCCCATGAGCGGCCATGCATGGAAGAGGTCTCAAGCACACGGACAAGAATAAGTCTATGCCT

ATCCGATGTTATATATCAAAATAAGTGCGT TTGGATGCATACA

Fig 6: PCR amplified DNA sequence

Query 1

GCGTACAGTTACCCCAAGGACGACGCCACCAGCATCTTCACTTGCCCCGGCGGCACAAAC 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 681

GCGTACAGTTACCCCAAGGACGACGCCACCAGCATCTTCACTTGCCCCGGCGGCACAAAC 740

Query 61

TACCAGGTCATCTTCTGCCCATGAGCGGCCATGCATGGAAGAGGTCTCAAGCACACGGAC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 741

TACCAGGTCATCTTCTGCCCATGAGCGGCCATGCATGGAAGAGGTCTCAAGCACACGGAC 800

Query 121

AAGAATAAGTCTATGCCTATCCGATGTTATATATCAAAATAAGTGCGTTTGGATGCATAC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 801

AAGAATAAGTCTATGCCTATCCGATGTTATATATCAAAATAAGTGCGTTTGGATGCATAC 860

Query 181 A 181

|

Sbjct 861 A 861

Fig 7: Amplified sequence blast result

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Sindhuri Madineni.

As the TLP’s have anti fungal and bacterial activity as per previous studies reported by [1], their influence on Candida

albicans 15,17,18, Saccharomyces cerevisiae 10 , Bacillus subtilis, Escherichia coli growth was tested using nutrient agar

media and the zone of inhibition were determined after 24 hr of incubation. The TLP protein had shown maximum

antimicrobial activity against Candida albicans when compared to other microorganisms. The zone of inhibitions for

each organism was shown in Fig. 8 and Table -1. TLP has no inhibitory activity against Escherichia coli, but the

activity on Candida albicans was maximum.

Table- 1: The zone of inhibitions for each organism

Candida albicans Bacillus subtilis

Saccharomyces

cerevesiae

E-coli

TLP protein +VE +VE +VE -VE

(a) Candida albicans

(b) Bacillus

(c) S.cerevesiae

CONCLUSION

Fig.8: Inhibition zones for Inhibition

Thaumatin like proteins have been extracted, purified and characterized from soaked and unsoaked grains of Hordeum

vulgare. The amount of protein in both the extracts (soaked and unsoaked) were determined and concluded that

protein concentration is high in soaked grains. The effect of pure proteins on the growth of microorganism has been

studied. It was found that TLP’s inhibited the growth of Candida albicans, Saccharomyces cerevisiae and Bacillus

subtilis. The TLP coding gene of 181bp length was amplified from the barley DNA and shown a 100% similarity with

the other TLP proteins.

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*Correspondence Author: Sindhuri Madineni ,

DVR & Dr.H.S. MIC College of Technology, Kanchikacherla, A.P, INDIA

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

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