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CLONING OF AN ANTIMICROBIAL HISTATIN DERIVED PEPTIDE BY RECOMBINANT
DNA TECHNOLOGY
Vishal Raval, Dipak Prajapati, Sandeep Patidar, Bijal Prajapati, Rajesh KS, Lalit Lata Jha
Parul Institute of Pharmacy, Waghodia, Limda, Vadodara-391760, India
ABSTRACT:
The purpose of the present research work is to clone an antimicrobial Histatin derived peptide by recombinant DNA technology.
Histatins are groups of twelve low molecular weight histidine rich peptides which are found in human submandibular and parotid
salivary secretions. They have shown to be potential antifungal agents when isolated and purified from human saliva. They are
also synthesized by chemical synthetic method but the cost of production of these antimicrobial peptides (AMPs) by synthetic
route is very high. The recombinant DNA technology pave a new way of producing histatin derived peptide in large amount and
cost of production will also be low. The recombinant DNA technology is the methodology in which an artificial gene can be
inserted into a vector and then the recombinant vector can be transformed inside the host cells. Typical vectors can be plasmid,
phage or cosmids and typical host cells used for transformations can be bacteria, yeasts, plants and animal tissues. One of the
prerequisite for expression of the peptide is that the plasmid vector should have the expression cassette constituting of promoter,
operator and other regulatory gene for synthesis of the protein inside the host cells for example E. coli. In this present work,
plasmid pBSKS is used to clone the Histatin derived peptide and Escherichia coli DH5α is used as the host cells. A 12 amino acid
fragment of histatin derived peptide was artificially synthesized and sequenced. This 12 amino acid fragment was then cloned
inside the pBSKS plasmid using enzyme Bam HI and Hind III. The recombinant pBSKS plasmid was them transformed inside
competent Escherichia coli DH5α host cells. The positive clones of E.coli DH5α were confirmed by Blue White colony screening
and Polymerase Chain Reaction method. Further the cloned plasmid can be transformed inside expression host cells like E.coli
BL21 and expression study of the peptide can be carried out using Poly Acrylamide Gel Electrophoresis (SDS PAGE) and
examining its biological activity against Candida albicans.
KEYWORDS: Histatin derived peptide, recombinant DNAtechnology, plasmid, host cells
Received on 11-04-2013
Modified on 21-06-2013
Accepted on 18-07-2013
are also cloned, produced and purified from E.coli cells for
drug design and discovery. The small DNA molecules are used
as a molecular probes which are used for disease diagnosis,
finger printing and as a therapeutic agents. The r DNA
technology is also used in gene delivery and gene therapy.
Monoclonal antibodies are also produced by this technology.2
INTRODUCTION:
Recombinant DNA technology, are used in DNA cloning,
which permits researchers to prepare large numbers of
identical DNA molecules. Recombinant DNA is simply any
DNA molecule composed of sequences derived from different
sources.1
Antimicrobial Peptides (AMPs) are the catalytic peptides
that are produced by the living organisms of all types
(vertebrates and invertebrates) including humans in response
to infection and exert antibiotic like action against pathogenic
micro organisms.3
Application of recombinant DNAtechnology:
Recombinant DNA technology is very useful technique for the
production of novel proteins in E. coli and yeast. Because of
this technique, vaccines and hormones are also produced in
bacteria, yeast, plants or animals. Human artificial receptors
Introduction and Classifications of Histatins:
Histatins are groups of twelve low molecular weight histidine
rich peptides which are found in human submandibular and
parotid salivary secretions. They are named as histidine 1-12,
out of which histatin 1, 3 and 5 are 38, 32 and 24 amino acid
Lalit Lata Jha
Parul Institute of Pharmacy, Limda, Vadodara, India.
Email: [email protected]
44
Cloning of histatin antimicrobial peptide
residues respectively. All other histatins varies from 7-38
amino acid residues in length. They are secreted by the salivary
glands and are one of the constituent of human saliva. In human
saliva concentration of histatins is found in the range of 50425μμ.4-5
Methods:
Comparison of Codon Bias table of human and E. coli
A graphical comparison between the Codon bias table of
human and E.coli was done and profound differences were
found in the codon usage of the two organisms. After
comparison, the best amino acid Codon which shows
maximum Codon usage in E.coli was selected. All the datas
were analysed by GCUA (Graphical Codon usage Analyser)
software and results were reported.
Histatin are showing fungicidal activity against a broad range
of pathogenic fungi including Candida albicans,
Cryptococcus neoformous and Asperigillus fumigates.6
The histatin exert its antifungal activity through the formation
of reactive oxygen species. histatins are taken up by the
Candida albicans cells and associate intra cellularly with
mitochondria and induces the formation of reactive oxygen
species which causes inhibition of respiration of mitochondria
and thereby to whole fungal cell.6
Designing of Histatin gene and Oligonucleotide primer for
PCR
The final selected Histatin gene with best codon usage in
E.coli was designed for directional cloning in pBSKS plasmid
by putting restriction enzyme sites Bam HI and Hind III. The
gene was also flanked by anchoring sequences required by
Bam HI and Hind III cutting sites. An initiation Codon ATG to
start the HTN peptide synthesis after restriction enzyme site of
Hind III was kept and also TGA which is the stop codon was
kept once the 12 amino acid sequence of Histatin derived
peptides ends. The total size of the Histatin derived peptide
was decided to be 66 base pairs long. The primers were
designed based on the Histatin gene sequence. 20 base pairs
forward and 20 base pair reverse primers were designed based
on the conserved sequence of the finally decided Histatin gene
sequences. These primers were used in the polymerase chain
reaction to amplify the cloned Histatin gene from the pUC
vector and for the identification of the cloned fragment. All the
decided Histatin gene base pair sequences and oligonucleotide
primers were sent to Amnion Life Sciences, Banglore for
synthesis and sequencing.
7
Therapeutic potential :
Histatins are shown to posses anticandidal activity. They are
broad spectrum antifungal agent mainly active against candida
albicans and pathogenic yeasts. Numbers of the histatin family
along with antifungal properties have shown to be major
wound closure stimulating factor and out of twelve Histatins,
Histatin 1 and 2 are especially shown to be wound healers.
Histatins can be used as an important component of artificial
saliva which is developed for treatment of salivary
dysfunction, topical vaccines against oral diseases and
diagnostic tests for local and systemic diseases. A synthetic
fragment of Histatin 5 which is 12 amino acid long shown to
retain anticandidal activity. The fragment is named as p-113
which can be used in treatment of oral candidiasis. The 12
amino acid fragment peptide is active against Candida
albicans, C.glabrata, C.parapsilosis and C.tropicalis. Some of
the report suggested that Histatins act synergistically with
commonly used antimycolics. The study shows dhvar 4, a
designed analogue of the human salivary peptide Histatin 5
shows strong synergism of Amphoterecin B against several
Aspergillus, Candida and Cryptococcus strains. Thus the
Histatin peptide are suitable for combination therapy. Another
synthetic peptide, muc-12 mer and Histatin5, 12 mer have
worked synergistically with Miconazole and Amphoterecin B.
They have shown synergistic activity against C.albicans and
C.neoformans. The toxicity test of individual compound was
also tested by haemolytic activity which shows very low level
of its toxicity. Many other synthetic analogues of Histatin
peptide indicated high antifungal therapeutic potential in
particular for the treatment of drug resistant fungal strains
associated with immune compromised patients. The same
peptide can also be used as components of artificial saliva for
patients with salivary dysfunction. Histatins are also shown to
prevent Closteridin Histolyticum infection by inhibiting
Clostripain, a proteinase enzyme formed in this pathogen.
They are also used in topical gel formulation, oral rinse
formulation and in dental acrylic.
Isolation of Plasmid8
A single colony of pBSKS plasmid harboring E.coli was
picked up and incubated into 10 ml of bacterial culture along
with 10μl of Ampicillin (100 mg/ml). Next day, the culture
was centrifuged at 4000 rpm for 10 min. The pellet was
resuspended at 400 μl of ice cold solution A (GTE buffer) and
transferred to 1.5 ml eppendorf's tube. The tube was incubated
for 10 min on ice. Then 800 μl of ice cold solution B (lysis
solution) was added and mixed well. Immediately after this
600 μl of ice cold solution C (genomic DNA precipitating
solution), was added and tapped gently. The tube kept in ice for
5 min and then centrifuged at 10,000 rpm for 10 mins. The
supernatant was then transferred to fresh eppendorf's tube.
Equal volume of isopropanol was added and kept at (-20°) for
2-3 hours to precipitate plasmid DNA. The pellet which was
obtained after centrifugation was washed with 70% ethanol
twice. The yield and purity of plasmid DNA was then
visualized byAgarose Gel Electrophoresis.
Agarose Gel Electrophoresis of Plasmid DNA9
For 1% gel.200 mg of Agarose was added to 20 ml of 0.5 X
Electrophoresis Buffer (EB).The Agarose was dissolved in
buffer by heating with constant swirling in microwave oven
until it dissolved completely. The gel cast was then taken and a
tape was wrapped around the two sides of it so as to form a
mold.A comb was inserted into position near one end of the gel
so that there is a 1 mm space between the bottom of the teeth
and the gel cast. When Agarose was cooled to about 50 to
60°C, it was slowly poured into the mold. After around half an
hour, when Agarose gets harden, the comb is carefully
MATERIALSAND METHODS:
Materials: Histatin, Agarose, Luria Bertani broth (LB), Tris
hydroxymethyl Aminomethane, Sodium lauryl Phosphate,
Luria Bertani Agar (LA), Ethyl Alcohol, Isopropanol, Boric
Acid, Dextrose Anhydrous, Glycerol, Sucrose, Potassium
Acetate.
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Cloning of histatin antimicrobial peptide
Then 2.5 ml of the grown culture was transferred in sterilized
Ria vials and were chilled in ice. After this the cells were
pelleted at 5000 rpn for 5 minutes at 4°C in refrigerated
centrifuge. The cells were then resuspend in 500 μl of pre
cooled 0.1M CaCl2.The tubes were kept in ice for 20 min and
then centrifuged at 5000 rpm for 5 minutes at 4°C.Finally the
pellet was resuspend in 100 μl of ice cold CaCl2 containing
20% glycerol in each Ria vials. All the competent cells were
stored at -20°C till further use.
removed from the gel and also slowly tape was also removed
from the sides of the gel cast. The gel was kept into the
electrophoresis apparatus and electrophoresis buffer (EB) was
added to fill reservoir and cover the gel with about 2mm of
buffer.DNA loading dye and DNA samples were mixed in a
transparent and clean plastic paper and loaded into the slots of
the submerged gel using a micropipette. Gel was then runned
for 90 minutes at 40 to 50 volts until the blue dye marker is near
the end of gel. After that the gel was soaked in a fresh solution
of ethidium bromide (10mg/ml) for 30 minutes for staining.
Then the gel was removed from staining solution and placed on
Benchtop UV Transilluminator and photographed.
Transformation of recombinant plasmid to competent
E.coli DH5α cells12
100 ng of plasmid was taken and added to 200 μl of competent
cells in an eppendorf's tube. A control was also kept without
plasmid. Both the tubes were then incubated in ice for 20 min.
Heat shock was given at 42°C for 90 secs in both the tubes and
immediately plunged in ice for 2-3 mins. Plasmid and
competent cells mixture was then added to 500 μl of Lb and
incubated at 37°C for 1 hour. The cells were pelleted at 4500
rpm for 19 min at 4°C.900 μl of supernatant was discarded and
pellet was gently resuspended on 100 μl of remaining
supernatant. The resuspended pellet was then plated on LA
plate with Ampicillin (100 μg/ml), X Gal and IPTG. The plate
was then incubated at 37°C for 18 hours for Blue White colony
screening.
Digestion of plasmid and Histatin gene with Hind III and
Bam HI10-11
Following reaction setup was done for digestion of plasmid
pBSKS:
pBSKS
10μl (1μg)
Buffer
X5μl
Hind III+Bam HI 2μl+2μl (40 units each)
Water
31μl
Total
50μl
Table 1: Digestion of plasmid
Following reaction set up was done for digestion of Histatin
gene:
Isolation of recombinant plasmid with Histatin derived
gene from transformed E.coli cells
The white colony from the previous LA plate with Ampicillin,
X Gal and IPTG were selected and inoculated into 10 ml of LB
medium along with 10 μl of Ampicillin (100mg/ml). Next day,
the overnight grown culture was centrifuged at 4000 rpm for
10 minutes. The pellet obtained was suspended 400 μl of ice
cold solution A (GTE buffer) and transferred to 1.5 ml
eppendorf's tube. The tube was incubated for 10 min on ice.
Then 800 μl of ice cold solution B (lysis solution) was added
and mixed well. Immediately after this 600 μl of ice col
solution C (genomic DNA precipitating solution),was added
and tapped gently. The tube kept in ice for 5 min and then
centrifuged at 10,000 rpm for 10 mins, the supernatant was
then transferred to fresh eppendorf's tube. Equal volume of
isopropanol was added and kept at -20°C for 2-3 hours to
precipitate plasmid DNA. The tube was again centrifuged at
10,000 rpm for 10 min. The pellet which was obtained after
centrifugation was washed with 70%ethanol twice. The yield
and purity of plasmid DNA was then visualized by Agarose
Gel Electrophoresis.
Histatin gene
10μl (1μg)
Buffer
(10 X)3μl
Hind III+Bam HI 2μl+2μl (40 units each)
Double distilled water
13μl
Total
30μl
Table 2: Digestion of Histatin gene
Ligation of plasmid vector and Histatin gene
The isolated Histatin gene and plasmid vector were purified by
HiPur Product Purification Spin Kit (MB512-20PR). Required
amount of the vector DNA and plasmid DNA were then
transferred to the sterile 0.5ml centrifuge. Required amount of
double distilled water is then added to this tube warmed to
45°C for 5 minutes to melt cohesive termini that may have co
annealed during fragment preparation. The DNA solution in
0.5 ml eppendorf's tube was then chilled to 0°C before the
remainder of the ligation reagents was added. The reaction
mixture was then incubated for 16 hours at 16°C. A 10μl
ligation reaction was set up as follows in table.
Polymerase Chain Reaction (PCR) to detect the presence
of Histatin derived peptide in plasmid pBSKS13-15
A polymerase chain reaction was set up for the detection of the
Histatin derived gene in recominant plasmid. Forward primer
and reverse primer which was supplied by the amnion life
sciences was dissolved in TE in the respective volumes to give
final concentration of 100 pmol/μl.
10 X Ligation Buffer
1.0μl
T4 DNA Ligase
0.5 μl(40 units)
Vector DNA
3.0 μl(100 ng)
Histatin DNA
2.0 μl (20 ng)
RO water
3.5 μl
Total
10 μl
Ingredients
Stock solution
(vol for 100 pmol/μl)
1 HTN 1 Forward primer
541
2 HTN 2 Reverse primer
545
Table 3: Ligation of plasmid with histatin gene
Preparation of Competent Cells of E.coli
Glycerol stock of E.coli DH5α was inoculated in 5 ml of LB
and grown overnight. Then 500 μl of overnight culture was
taken from above and inoculated into 50ml of LB. The cells
were allowed to grow till optical density reaches to 0.3 to 0.4;
Table 4: Primers used for PCR
46
Cloning of histatin antimicrobial peptide
The stock solutions of primers were further diluted in the ratio 1:10 to give a final concentration of 10pmol/μl. Four different
polymerase reactions were carried out by using different concentration of forward primers and reverse primer in the range of 0.5
pmol/μl. The annealing temperature was kept at 60°C and extention was carried out 72°C.A20 μl volume reaction was set up.
Table 5: PCR with different concentration of primers
16
Detection and visualization of PCR amplified band
The entire four PCR product obtained was loaded in 2.5% Agarose gel casted on a gel electrophoresis unit. The PCR samples were
mixed with DNA loading dye in a transparent and clean plastic paper and loaded into the slots of the submerged gel using a
micropipette. Gel was then runned for 60 minutes at 40 to 50 volts until the blue dye marker is near the end of gel. After that the gel
was soaked in a fresh solution of Ethidium bromide (10mg/ml) for 30 minutes for staining. Then the gel was removed from
staining solution and placed on Benchtop UV transilluminator and photographed.
RESULTS & DISCUSSION:
Comparison of codon bias table of human and E.coli
From the graph it can be shown that the Codon for the amino acidArginine, CGT was 100% used in human but only 28% used in E.
coli systems. Similarly other Codon systems were tested and compared. The best Codon base pairs usage of the species, which is
highly used in E. coli was selected for one particular amino acid.
47
Cloning of histatin antimicrobial peptide
Fig1: comparison of codon table of human and E.coli.
Designing of Histatin gene and Oligonucleotide primers for PCR:
Based on the comparison of Codon bias table the best gene sequence which will show expression in E. coli was selected. Similarly
the Oligonucleotide primers were synthesized for Polymearse Chain Reaction (PCR). After designing of the Histatin gene and the
Oligonucleotide primers both the synthesis and sequencing were carried byAmnion Life Sciences, Banglore.
Fig.2: Sequencing of gene
48
Cloning of histatin antimicrobial peptide
Fig.3: Sequencing of Gene
Isolation of plasmid
From 50 ml of plasmid harbouring E. coli cells, around 1 μg of
plasmid DNA was obtained which was sufficient to carry out
the further cloning experiment. Plasmid isolation by alkaline
detergent method is a rapid method for isolation of plasmid
DNA. There is first resuspension of pellet in solution A (GTE
buffer) which gives much better lysis than the direct treatment
of pelleted cells. By the alkaline SDS lysis step (Solution B)
linear and chromosomally DNA are selectively denatured,
while covalently closed circular DNA (plasmid) are not
affected. Proteins are also denatured at pH 12.5 thereby
reducing the possibility of enzymatic degradation of plasmid
DNA. The SDS lyse the cells and denature the proteins. During
neutralization step (Solution C), pH of the mixture is lowered
around 4.8 which causes chromosomal DNA to form an
insoluble complex that together with protein SDS complex is
precipitated by the high salt concentration of potassium
acetate. Finally the plasmid DNA was precipitated from
supernatant by adding two volumes of ethanol and keeping
overnight at -200C. The pellet may or may not be visible after
centrifugation depending upon the purity of the plasmid DNA.
Agarose Gel Electrophoresis of plasmid DNA
Fig.4: Agarose Gel Electrophoresis
49
Cloning of histatin antimicrobial peptide
The following picture shows the quality of plasmid DNA, pBSKS and its purity along with the Histatin gene.
Fig.5: Isolation of plasmid and Histatin gene
Preparation of competent cells of E. Coli. DH5α along
with control
Digestion of pBSKS plasmid and Histatin gene with
HindIII and Bam HI
For cloning both the plasmid pBSKS and Histatin gene were
digested by restriction enzymes like Bam HI and Hind III and
found to be o.k. The following diagram shows the digestion of
plasmid as well as Histatin gene.
Following photograph shows the competent cells prepared
with white colonies in figure no 8 along with control.
Digested Histan gene
Lane 1
HTN
gene
Lane 2
PCR
marker
Fig 6: Digestion of plasmid and Histatin gene
Fig. 8: Competent cells and Control
Fig.7: Ligation of plasmid Vector and Histatin gene in
Ligation Bath
50
Cloning of histatin antimicrobial peptide
Transformation of recombinant plasmid to competent
E.coli DH5α (Blue White colony screening)
Fig.9: Blue White colony screening
Isolation of recombinant pBSKS plasmid with Histatin
derived gene from transformed E. coli cells
From 50 ml of recombinant pBSKS harboring plasmid with
Histatin gene, around 1 μg of plasmid DNA was obtained
which was sufficient to carry out the further PCR
amplification.
Fig.11: Amplified bands by PCR
CONCLUSION:
Histatin derived peptides can be produced in large quantities
by recombinant DNA technology. The synthetic artificial gene
of Histatin was successfully inserted into pBSKS vector and
then the recombinant vector was transformed inside the host
cell, Escherichia coli DH5α. The positive clones of E.coli
were confirmed by Polymerase Chain Reaction. Further the
recombinant plasmid can be transformed inside E. coli BL21
cells to check the expression studies of the peptide by SDS
PAGE and biological activity of the peptide against C.
albicans. The purified peptide can be used as a potential drug
in treatment of oral infection in the form of mouth rinse
formulation or in control of skin infections.
Polymerase Chain Reaction (PCR) to detect the presence of
Histatin derived peptide in the plasmid pBSKS
ACKNOWLEDGEMENT:
The authors are thankful to management of Parul Group of
Institutes for providing necessary infrastructure and chemicals
to carry out the research work.
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