Nov.2011-Jan.2012. Vol.2.No.1, 273-278 E- ISSN: 2249 –1929
Journal of Chemical, Biological and Physical Sciences
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
DVR & Dr.H.S. MIC College of Technology, Kanchikacherla, A.P, INDIA
Received: 18 November 2011; Revised: 26 November 2011; Accepted: 30 November 2011
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
Keywords: Thaumatin-like protein, Pathogenesis-Related protein, Barley, Antimicrobial activity.
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
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
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-
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
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’-
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
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.
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
275 J. Chem. Bio. Phy. Sci. Sec. B, Nov. 2011- Jan. 2012, Vol.2, No.1, 273-278
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.
Fig 6: PCR amplified DNA sequence
Query 181 A 181
Sbjct 861 A 861
Fig 7: Amplified sequence blast result
276 J. Chem. Bio. Phy. Sci. Sec. B, Nov. 2011- Jan. 2012, Vol.2, No.1, 273-278
As the TLP’s have anti fungal and bacterial activity as per previous studies reported by , 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
TLP protein +VE +VE +VE -VE
(a) Candida albicans
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.
1. S. Garjanovic, M.V. Beljanski, M. Gavrovic-Jankulovic, G. Gojgic-Cvijovic, M.D. Pavlovic and F. Bejosano,
J.Inst.Brew. 2007, 113(2) 206.
2. A. Edreva. Gen.Appl.Plant Physiology, 2005, 31 (1-2), 105.
3. L.C. Van Loon, M. Rep and C.M.J. Pieterse, Annu.Rev. Phytopath, 2006, 44, 135.
4. H. Breitender, Thaumatin-like proteins—a new family of pollen and fruit allergens, Allergy 2004,59, 479.
277 J. Chem. Bio. Phy. Sci. Sec. B, Nov. 2011- Jan. 2012, Vol.2, No.1, 273-278
5. A. Stinzi, T. Heitz, V. Prasad, S. Wiedmann-Merdinohlu, S. Kauffman, P.Geoffry, M. Legrand and B. Fritig,
Biochemie.1993, 75, 687.
6. L.C. Van Loon and E.A. Van Strien, Phys.Mol.Plant Path, 1999. 55, 85.
7. S.Muthukrishnan, H.George Liang, Harold N.Trick & S. Bikram Gill, Plant Cell, Tissue & Organ Culture.
2001, 64, 93.
8. E. Reiss, and C. Horstman, Physol.Mol.Plant.Pathol. 2001, 58, 183.
9. J Hejgaard, S Jacobsen, I Svendsen. FEBS Letters, 1991, 291, 1, 127.
10. T, Jean, G. Jean., P. Claude and A. Asselin. Plant Physiol. 1998, 118, 1431.
11. S. Garjanovic, J.Inst.Brew. 2009, 115 (4), 334.
12. E. Fierens, K. Gebruers, AR. Voet, M. De Maeyer, C.M. Courin, J.A. Delcour, J Enzyme Inhib Med chem.
13. E. Fierens, S. Rombouts, H. Goesaert, K. Brijs, J. Beaugrand, G. Volckaert, S. Van Campenhout, P. Proost,
C.M. Courtin and J.A. Delcour. Biochem. J. 2007, 403, 583.
14. V.Barod, S.Sriram. Asian. J.Exp.Sci, 2008, 22, 3, 189.
15. R.I.W.Osmond, Maria H, Fabien F, Anne I and Goeffrey B. fincher. Binding interactions between barley
thaumatin-like proteins and (1, 3)-beta-D-glucans kinetics, specificity, structural analysis and biological
implications Eur. J. Biochem. 2011, 268, 4190.
16. J. Chou, Y Huang Differential expression of thaumatin-like proteins in sorghum infested with green bugs.
Journal of Biosciences.2010, 65, 0939-5075.
17. Subramanian. J, Zhijian Li and D.J.Gray. Constitutive Expression of Vitis viniferra Thaumatin like protein
after invitro selection and its role in antharacnose resistance. Functional Plant Biology, 2003, 30, 1105.
18. A.J. Vigers, S. Wiedemann, K.R. Walden., M. Legrand, Claude P. Selirennikoff and B. Fritig. Thaumatin-like
pathogenesis-related proteins are antifungal. Plant Science. 1992, 83, 2, 155.
19. J Hejgaard, S Jacobsen, I Svendsen. FEBS Letters. 1991, 291, 1, 127.
*Correspondence Author: Sindhuri Madineni ,
DVR & Dr.H.S. MIC College of Technology, Kanchikacherla, A.P, INDIA
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