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CONSTRUCTION AND EXPRESSION OF A SYNTHETIC

ANTIMICROBIAL PEPTIDE IN Escherichia coli

A PROJECT REPORT
Submitted by
SAISHREYAS GOURISHANKAR IYER

In partial fulfillment for the award of the degree


Of
BACHELOR OF TECHNOLOGY
IN
BIOTECHNOLOGY

SRI VENKATESWARA COLLEGE OF ENGINEERING,


SRIPERUMBUDUR, Tk - 602117

ANNA UNIVERSITY: CHENNAI 600 025


APRIL 2017

1
ANNA UNIVERSITY: CHENNAI 600 025

BONAFIDE CERTIFICATE

Certified that this project report titled “CONSTRUCTION AND EXPRESSION


OF A SYNTHETIC ANTIMICROBIAL PEPTIDE IN Escherichia coli” is the
bonafide work of “SAISHREYAS GOURISHANKAR IYER” (Reg No:
212713214028) who carried out the project work under my supervision.

SIGNATURE SIGNATURE
Prof. E. NAKKEERAN Ms.N.KANAGAM
HEAD OF THE DEPARTMENT SUPERVISOR
PROFESSOR ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY DEPARTMENT OF BIOTECHNOLOGY
SRI VENKATESWARA COLLEGE OF SRI VENKATESWARA COLLEGE OF
ENGINEERING, SRIPERUMBUDUR, ENGINEERING, SRIPERUMBUDUR,
TK – 602117 TK - 602117

Submitted for the project viva voce examination held on _______________

INTERNAL EXAMINER EXTERNAL EXAMINER

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ACKNOWLEDGEMENTS

I would like to take this opportunity to thank each and every one who had helped me
throughout the entire project in making great success.
On the forefront, I would like to extend my gratitude to Prof. M. Sivanandham,
Secretary and Visiting Professor of Biotechnology, Sri Venkateswara College of
Engineering for providing the support.
I would like to thank our Principal of Sri Venkateswara College of Engineering,
Prof. S. Ganesh Vaidyanathan for giving me the opportunity to do my project in the
college.
I sincerely thank Prof. E. Nakkeeran, Head of the Department of Biotechnology for
individually looking through my project.
It would be incomplete if I do not thank my project guide, Ms. N. Kanagam,
Assistant Professor, Department of Biotechnology, for monitoring the project
completely and for guiding me the right way without which this project would not
have been possible.
I would like to thank Prof. Nalinkanth. V. Ghone, Professor, Department of
Biotechnology, for all his advice and support.
I would also like to thank Mr. J. Hariharan, Assistant Professor, Department of
Biotechnology, for his invaluable help with genetic engineering work.
I would like to extend my sincere gratitude to all the other faculty members and lab
assistants for their valuable ideas and help. Finally, I would like to thank my parents
and friends for their encouragement and cooperation.
Saishreyas Gourishankar Iyer

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ABSTRACT

Antimicrobial peptides have existed as a part of the innate immune response of all

classes of life for millennia. Yet even today it remains one of the new and under-

researched areas of biology. Though detailed study of a few well known

antimicrobial peptides can give us valuable insights into its structure, function,

mechanism of action and targets, a more broad minded study is also required to

gain a deeper understanding into the science behind these remarkable molecules.

The present study has focused on the designing of antimicrobial decapeptides

consisting of alternating amino acid residues. 175 candidates were initially

proposed. Online and offline prediction and modelling tools were used in the

designing process. A three tier process was carried out, each tier following a

different method of predicting antimicrobial activity. Finally, 14 of these peptides

were selected as good antimicrobial candidates. Following this comparison, these

candidate molecules were modelled and cluster calculation was done for each of

them using liquid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as

solvent. One of these peptides, N – IWIWIWIWIW – C, was predicted to be a

signal peptide, while N – KLKLKLKLKL – C and its reverse were predicted to

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have very good anticancer properties in addition to being good antimicrobial

candidates. The peptide having arginine and tryptophan, one of the final 14, was

selected for proceeding to wet laboratory studies based on previous evidence of its

antimicrobial activity. A comparison was made among random peptides, all having

Arg and Trp and N - MWRWRWR- C was selected as the best. A plasmid

construct was designed using lac promoter and pSB1C3 backbone. In the future,

transformation and expression of this plasmid in E.coli will be done. This will be

followed by testing the antimicrobial effectiveness of the peptide against a range of

microorganisms.

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TABLE OF CONTENTS

Chapter Title Page


no. no.

ABSTRACT i

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS AND SYMBOLS ix

1 INTRODUCTION 1

1.1 ANTIMICROBIAL PEPTIDES 1

1.2 PREVIOUS WORK DONE 3

1.3 THE PRESENT STUDY 4

2 REVIEW OF LITERATURE 6

3 AIM AND OBJECTIVE 10

4 MATERIALS AND METHODS 11

6
4.1 IN SILICO STUDIES 11

4.1.1 The designing of the antimicrobial peptides 11

4.1.2 Antimicrobial index for each decapeptide 13

4.1.3 Presence of hydrophobic amino acids 14

4.1.4 CAMP R3 (Collection of Anti Microbial 15


Peptide) prediction

4.1.5 SignalP 4.1 server prediction 17

4.1.6 Anticancer properties 18

4.1.7 Secondary structure prediction 18

4.1.8 Selection of antimicrobial peptide for 20


laboratory work

4.2 LABORATORY WORK 20

4.2.1 Restriction 21

4.2.2 Ligation 22

4.2.3 Agarose Gel Electrophoresis 23

4.2.4 Competent cell preparation 25

4.2.5 Transformation 27

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4.2.6 Plasmid DNA isolation 28

5 RESULTS AND DISCUSSION 30

5.1 IN SILICO STUDIES 30

5.1.1 The first tier elimination 30

5.1.2 The second tier elimination 32

5.1.3 The third tier elimination 32

5.1.4 Is it a signal peptide? 33

5.1.5 Anticancer properties 34

5.1.6 Secondary structure prediction 34

5.1.7 Selection of antimicrobial peptide for 45


laboratory work

5.1.8 Plasmid design 46

5.2 LABORATORY WORK 47

5.2.1 Plasmid construction 47

5.3 DISCUSSION 48

6 CONCLUSION 50

7 REFERENCES 51

8
LIST OF TABLES

Table No Description Page No


1 Antimicrobial index of amino acid residues 12
2 The comprehensive table for the 14 decapeptides 35

3 The comprehensive table for the 14 decapeptides 36

4 Comparison of random peptides having arginine and 46


tryptophan

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LIST OF FIGURES

Fig No Description Page


No
1 Various structures of antimicrobial peptides 2

2 Mechanism of action of antimicrobial peptides 2

3 The sachet system designed by iGEM 2016 3

4 The APD3 Antimicrobial Peptide Calculator and Predictor tool 14

5 The CAMP website showing the tools available for predicting 16


antimicrobial peptides

6 The CAMP website showing the “Predict Antimicrobial 16


Peptides” tool

7 SignalP 4.1 server 17

8 The AntiCP server for predicting anticancer peptides 18

9 The Ascalaph Designer software 19

10 The Vega ZZ Molecular Modelling software 20

11 Mean antimicrobial indices of decapeptides consisting of two 31


alternating amino acid residues

10
12 SignalP 4.1 Results 33

13 N-RCRCRCRCRC-C 38

14 N-KCKCKCKCKC-C 38

15 N-WRWRWRWRWR-C 39

16 N-RWRWRWRWRW-C 39

17 N-KWKWKWKWKW–C 40

18 N-WKWKWKWKWK-C 40

19 N-IKIKIKIKIK-C 41

20 N-IKIKIKIKIK-C 41

21 N-FKFKFKFKFK–C 42

22 N-KLKLKLKLKL–C 42

23 N-LKLKLKLKLK–C 43

24 N-CCCCCCCCCC–C 43

25 N-WWWWWWWWWW-C 44

26 N-IWIWIWIWIW-C 44

27 Plasmid design for the lac AMP cassette 47


28 The antimicrobial peptide cassette viewed in a gel. 48

11
LIST OF ABBREVIATIONS AND SYMBOLS

Abbreviations, Expansion
nomenclature
Gly (G) Glycine
Ala (A) Alanine
Leu (L) Leucine
Met (M) Methionine
Phe (F) Phenylalanine
Trp (W) Tryptophan
Lys (K) Lysine
Gln (Q) Glutamine
Glu (E) Glutamic Acid
Ser (S) Serine
Pro (P) Proline
Val (V) Valine
Ile (I) Isoleucine
Cys (C) Cysteine
Tyr (Y) Tyrosine
His (H) Histidine

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Arg (R) Arginine
Asn (N) Asparagine
Asp (D) Aspartic Acid
Thr (T) Threonine
CAMP Collection of Anti Microbial Peptides
AMP Antimicrobial peptide
POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine
APD Antimicrobial Peptide Database
AI Antimicrobial Index
WWH Wimley White Whole Residue
Hydrophobicity
BI Bowman Index
EC Extinction Coefficient
MR Mammalian Reticulocytes
GRAVY Grand Average of Hydropathicity
SVM Support Vector Machine
RFC Random Forest Classifier
ANN Artificial Neural Network
DAC Discriminant Analysis Classifier
RBS Ribosome Binding Site
Lac Lactose

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E.coli Escherichia coli
µl Microlitre
mM Millimolar
ng Nanogram
° Degree
°C Celsius
EDTA Ethylenediaminetetraacetic acid
DNA Deoxyribonucleic acid
OD Optical Density
UV Ultraviolet
V Volt
TE Tris EDTA
TBE Tris Borate EDTA
BSA Bovine Serum Albumin
ATP Adenosine triphosphate
NaOH Sodium hydroxide
SDS Sodium dodecyl sulphate
EtBr Ethidium bromide
CaCl2 Calcium chloride
MnCl2 Manganese chloride
MgCl2 Magnesium chloride
H2O Water

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NaCl Sodium chloride
KCl Potassium chloride
MgSO4 Magnesium sulphate

15
CHAPTER 1

INTRODUCTION

1.1 : ANTIMICROBIAL PEPTIDES

Antimicrobial peptides (Host Defense Peptides) are small molecular weight


proteins with broad spectrum antimicrobial activity against bacteria, viruses, and
fungi. They are a part of the innate immune response found among all classes of
life. Antimicrobial peptides are finding a lot of applications in a variety of fields.
Synthetic peptides could even replace conventional antibiotics because of their
specificity, speed and because bacteria cannot easily develop resistance towards
them. They have been demonstrated to kill Gram negative and Gram
positive bacteria, enveloped viruses, fungi and transformed or cancerous cells
(Reddy et al., 2004). Unlike the majority of conventional antibiotics it appears as
though antimicrobial peptides may also have the ability to enhance immunity by
functioning as immunomodulators. Antimicrobial peptides are generally between
12 and 50 amino acids. These peptides include two or more positively charged
residues provided by arginine, lysine or, in acidic environments, histidine, and a
large proportion (generally >50%) of hydrophobic residues. The secondary
structures of these molecules follow 4 themes, including i) α helical, ii) β stranded
due to the presence of 2 or more disulfide bonds, iii) β-hairpin or loop due to the
presence of a single disulfide bond and/or cyclization of the peptide chain, and iv)
extended.

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Figure 1 : Various structures of antimicrobial peptides

This image shows the 4 main classes of antimicrobial peptides with examples.(source :
en.wikipedia.org/wiki/Antimicrobial_peptides#/media/File:Various_AMPs.png)

Figure 2 : Mechanism of action of antimicrobial peptides

There are many proposed modes, the main ones being formation of a pore in the membrane and
penetration(source:en.wikipedia.org/wiki/Antimicrobial_peptides#/media/File:Modes_of_action.
png)

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1.2 : PREVIOUS WORK DONE

A major problem faced by the medical world is the problem of antimicrobial


resistance. Many scientists are turning towards synthetic peptides that are more
specific and potent than their natural counterparts. One such class of peptides is the
short Cationic Antimicrobial Peptides (scAMPs) (Wenzel et al., 2014). A study by
Wenzel et al., (2014) showed that scAMPs made of arginine and tryptophan had
potent antimicrobial activity. They showed that these peptides killed many Gram
positive and Gram negative organisms by delocalization of peripheral membrane
proteins. The positive charge of arginine and the lipophilic nature of tryptophan
were shown to be responsible for the antimicrobial activity.

The SVCE CHENNAI team of iGEM 2016 did a project that could eradicate the
problem of milk spoilage. They designed a sachet system for delivering AMPs
into the milk for killing the microorganisms present in milk.

Figure 3 : The sachet system designed by iGEM 2016

Two small cationic antimicrobial peptides (cAMPs) were designed; both have

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alternating arginine and tryptophan. One cAMP has been designed to have two
glycine residues at the N terminal to increase its half life.
• Peptide 1: MWRWRWR (‘WR 1‘– SVCE iGEM 2016)
• Peptide 2:MGGWRWRWR (‘WR 2’ – SVCE iGEM 2016)

These cAMPs kill the bacterial cells through different mechanisms that depend
mainly on the unique properties of arginine and tryptophan. Guanidinium group of
arginine forms bidentate hydrogen bonds with negatively charged phosphate,
sulfate and carboxylate groups on the cell surface. The aromatic side chain in
tryptophan, helps it establish π- anion interactions with the sulfate groups of GAGs
in the bacterial cell wall, allowing the peptide to insert itself into the cell wall.
Two separate constructs were designed to make the process of AMP production
temperature controlled – one using an RNA thermometer and the other using a λpR
promoter.
1.3 : THE PRESENT STUDY
Every project done on AMPs takes us one step closer to understanding the science
behind these remarkable proteins.
The present study focuses on the following
1. To theoretically design and validate antimicrobial peptides using bioinformatics
tools.
2. To come up with a few best AMPs from among all of the ones designed.
3. To model each of the AMPs and to do cluster calculation using Liquid POPC as
solvent.
4. To test one of the designed AMPs in the laboratory.

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The present study studies the effect of alternating amino acid residues on the
antimicrobial index of the peptide in which they are present. It explores the
possibility of very small peptides being potential AMP candidates. It shows us that
two peptides with the same amino acid composition need not have the same
properties.

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CHAPTER 2

REVIEW OF LITERATURE

A lot of research has been done in the field of antimicrobial peptides, but still, not
a lot is known about their mechanism of action, their target and their potency. A lot
of web servers are available that offer tools for predicting antimicrobial regions in
the query peptide. But every website bases its prediction on a different set of facts.
The present study focuses on designing antimicrobial peptide candidates and
testing at least one of the designed peptides in the lab for efficiency. The designing
process has been done using 3 of the best antimicrobial peptide prediction tools
available online, each one basing their prediction on a different set of valid facts.

This has been done to get an even more accurate prediction of the potential
antimicrobial peptide candidates.

The first prediction tool used is AMPA Antimicrobial Sequence Scanning System.
The authors have used the antimicrobial peptide bactenecin 2A for their study in
which they have found its IC50 values for every one of its amino acid
replacements. They have come up with an antimicrobial index for every amino
acid that gives a fair assessment of the tendency of any amino acid to be found
within the AMP sequence.

Torrent et al., (2012), have concluded that AMPA-derived AI values can be used to
automatically classify proteins or domains as either antimicrobial or non-

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antimicrobial. They have said “The algorithm has been extensively validated in
silico and found to correctly identify 80–90% of the antimicrobial proteins and
properly predict their antimicrobial domain.”

The second level of prediction was done using APD3 server which predicts
antimicrobial peptides based on whether the peptides have hydrophobic residues
and what their net surface charge is. It also compares the peptide with all the other
antimicrobial peptides in its database.

Wang et al., (2015), have improved the peptide prediction interface in APD3. It
now predicts the possibility of a sequence to be AMPs based on the entire
parameter space defined by all the natural peptides registered in the database.
The third level of prediction was done using CAMP - Collection of Anti Microbial
Peptides server’s prediction tool which uses Artificial Neural Network,
Discriminant Analysis, Support Vector Machine and Random Forest Classifier to
compare the query peptide to all the peptides in their database.
Waghu et al., (2016), have based their tool on the fact that antimicrobial peptides
(AMPs) are known to have family-specific sequence composition, which can be
mined for discovery and design of AMPs. CAMP is a database of sequences,
structures and family-specific signatures of prokaryotic and eukaryotic AMPs.
CAMPR3 presently holds 10247 sequences, 757 structures and 114 family-specific
signatures of AMPs. Users can use the sequence optimization algorithm for
rational design of AMPs. The database integrated with tools for AMP sequence
and structure analysis will be a valuable resource for family-based studies on
AMPs.

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Tyagi et al., (2013), that usually Cys, Gly, Ile, Lys, and Trp are dominated at
various positions in anticancer peptides. They developed support vector machine
(SVM) based models using various features of peptides like amino acid
composition, dipeptide composition and binary profile pattern In addition, models
discriminating ACPs from AMPs have also been developed by them. The
prediction tool gives the output as an SVM score. The higher the score, the better.
Wenzel et al., (2014), have tested the antimicrobial effects of a hexapeptide having
alternating arginine and tryptophan residues on a range of microorganisms and
have come to the conclusion that it is a potential antimicrobial candidate. Their
study shows that the positive charge of arginine and the hydrophobic nature of
tryptophan contribute significantly to the antimicrobial property of the protein they
are part of.

Petersen et al., (2011), have used three types of scores, C, S and Y scores to predict
signal peptide regions within proteins.

C-score distinguishes signal peptide cleavage sites from everything else.

The C-score is trained to be high at the position immediately after the cleavage
site.

S-score (signal peptide score) distinguishes positions within signal peptides from
positions in the mature part of the proteins and from proteins without signal
peptides.

Y-score (combined cleavage site score)is combination (geometric average) of the


C-score and the slope of the S-score, resulting in a better cleavage site prediction
than the raw C-score alone. The Y-score distinguishes between C-score peaks by

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choosing the one where the slope of the S-score is steep.

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CHAPTER 3

AIM AND OBJECTIVE

Aim : To design, construct and express an antimicrobial peptide in Escherichia


coli.

Objective :

1. To design several antimicrobial peptides and to compare their physical, chemical


and antimicrobial properties.

2. To select a few peptides with the best antimicrobial properties, to model them
and to perform cluster calculation for these peptides using Liquid POPC as solvent.

3. To express an antimicrobial peptide in E.coli and then test it against a range of


microorganisms.

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CHAPTER 4

MATERIALS AND METHODS

4.1 : IN SILICO STUDIES

4.1.1 : The designing of the antimicrobial peptides.

The antimicrobial indices given in the AMPA Antimicrobial Sequence Scanning


System (Torrent et al., 2012) was used to design only a limited number of peptides
based on the antimicrobial indices of the individual amino acids. The AMPA
algorithm uses an antimicrobial propensity scale to generate an antimicrobial
profile by means of a sliding window system. The propensity scale has been
derived using high-throughput screening results from the AMP bactenecin 2A, a
12-residue peptide for which antimicrobial IC50 values for all amino acid
replacements at each position have been determined. From the IC50 for each
substitution, an antimicrobial index for individual residues can be calculated (Table
1) that provides a fair assessment of the tendency of such amino acid to be found
within an AMP sequence.

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Table 1 : Antimicrobial index of amino acid residues

The present study is based on the top 5 amino acids in this table. (source : AMPA website)

Residue Arg Lys Cys Trp Tyr Ile Val

PV 0.106 0.111 0.165 0.172 0.185 0.198 0.200

Residue His Asn Thr Phe Leu Gln Gly

PV 0.202 0.240 0.242 0.246 0.246 0.248 0.265

Residue Met Ser Ala Pro Glu Asp

PV 0.265 0.281 0.307 0.327 0.449 0.479

As low IC50 values correspond to high activity, amino acids with a low
antimicrobial index are the most favored to be part of an AMP. Each peptide
consisted of two alternating amino acid residues to give a total of ten residues for
each peptide. The amino acids with the top five antimicrobial indices according to
the AMPA website were selected for this purpose. These are –

Arginine – 0.106

Lysine – 0.111

Cysteine – 0.165

Tryptophan – 0.172

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Tyrosine – 0.185

The lower the antimicrobial index of an amino acid, the better the candidate for
designing an antimicrobial peptide (AMP). (Torrent et al., 2009) Decapeptides
consisting of the above listed amino acids, each, combined with all the other 20
amino acids were written down along with the reverse peptide for each peptide
designed. The total came to 175 decapeptides.

4.1.2 : Antimicrobial index for each decapeptide

All the decapeptides were run in the AMPA website tool to determine the mean
antimicrobial index of each peptide. The window was set as 5 and the threshold as
0.225. The peptides were ranked according to the results, with the peptides with
the lowest mean antimicrobial indices being considered the best. It was observed
that the reverse decapeptide for any decapeptide always had the same mean
antimicrobial index, even though, maximum and the minimum antimicrobial index
for every amino acid within the peptide showed variation between the two
peptides. Only those decapeptide with an index below 0.24 were selected for the
next step. Only 80 decapeptides made it through this process.

AMPA website predicts antimicrobial property based only on the antimicrobial


index. But to create really sound antimicrobial peptides, using this sorting process
alone wasn’t enough. The antimicrobial property of any AMP depends not only on
whether the constituent amino acids have low antimicrobial index but also on the
physical and the chemical properties of these amino acids. One such property that
appears to be crucial to antimicrobial activity is the hydrophobicity of the amino

28
acids.

It has been observed that hydrophobic amino acids contribute significantly to the
antimicrobial activity of any AMP they are part of. (Wang and Wang., 2004;
Wenzel et al., 2014).

4.1.3 : Presence of hydrophobic amino acids

The Antimicrobial Peptide Database (APD) contains 2830 antimicrobial peptides


from six kingdoms (294 bacteriocins and peptide antibiotics from bacteria, 4 from
archaea, 8 from protists, 13 from fungi, 341 from plants, and 2116 from animals).
It also has an “Antimicrobial Peptide Predictor and Calculator” tool.

Figure 4 : The APD3 Antimicrobial Peptide Calculator and Predictor tool

It gives the mean antimicrobial index of the query peptide. (source: APD3 website)

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This tool was used to find the Wimley White Whole Residue Hydrophobicity and
the Bowman Index for all the peptides. In the APD3 Antimicrobial Peptide
Database website, the Peptide design -> Improve Peptide options were selected and
each of the 80 peptides were entered as query. Based on the above observation, all
the decapeptides that did not contain any hydrophobic residues were eliminated.

4.1.4 : CAMP R3 (Collection of Anti-Microbial Peptides) prediction

The CAMP website AMP prediction tool was used. This tool has 4 different
outputs.

1. Support Vector Machine (SVM)


2. Random Forest Classifier (RFC)
3. Artificial Neural Network (ANN)
4. Discriminant Analysis Classifier (DAC)

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Figure 5 : The CAMP website showing the tools available for predicting antimicrobial
peptides. (source : CAMP website)

Figure 6 : The CAMP website showing the “Predict Antimicrobial Peptides” tool.

(source : CAMP website)

31
All 4 outputs were considered and only the peptides which scored a maximum
positive result in at least 3 output formats were selected. (Waghu et al., 2015)

4.1.5 : SignalP 4.1 server prediction


One of the most important properties of antimicrobial peptides that are expressed
by any host organism is their ability to exit the host cell and interact with the target
cell. While their interaction with the target cell is determined mostly by surface
charge (Wenzel et al., 2014), their ability to leave the host cell depends on
presence of specific signal peptide sequences within the peptide. (Petersen et al.,
2011).Whether the designed peptides could be signal peptides or could contain a
signal peptide or not was predicted using the SignalP 4.1 Server.

Figure 7 : The SignalP 4.1 Server.

(source: SignalP 4.1 server)

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4.1.6 : Anticancer properties
To test whether the 14 decapeptides had any anticancer properties, the AntiCP
website was used.(Tyagi et al., 2013)

Figure 8 : The AntiCP server for predicting anticancer peptides


(source : the AntiCP website)

4.1.7 : Secondary structure prediction


To predict the secondary structure of the peptides, the PEP-FOLD 3 de novo
peptide structure prediction site was used (Lamiable et al., 2016) The predicted
structure was then fed as pdb files into Ascalaph Designer. The “short optimization
to remove atomic clashes” option was used to do Energy Minimization.

33
Figure 9 : The Ascalaph Designer software
(source : The Ascalaph Designer software)

The Energy Minimized structures were then fed into Vega ZZ software to visualize
the 3D structure of the peptides in tube form. Vega ZZ is the evolution of the well
known VEGA OpenGL package and includes several new features and
enhancements. VEGA was originally developed to create a bridge between most of
the molecular software packages only, but in the years, enhancing its features, it's
evolved to a complete molecular modeling suite. This software is FREE for non-
profit academic uses.

Then cluster calculation was performed on each of the peptides with the solvent
set as POPC-LIQUID, thickness = 20Å, overlap = 0.8Å, type = layer and cluster
position as geometry center.
POPC is a chemical compound. It is a diacylglycerol and phospholipid. The full
name is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. It is an important

34
phospholipid for biophysical experiments and has been used to study various
subjects such as lipid rafts.

Figure 10 : The VEGA ZZ Molecular Modelling software


(source : Vega ZZ software)

4.1.8 : Selection of antimicrobial peptide for laboratory work


The AMP having arginine and tryptophan was selected for lab work. A comparison
was made among random peptides all having arginine and tryptophan.

4.2 : LABORATORY WORK (Ausubel, F.M., 1999)

35
4.2.1 : Restriction
Principle :
A restriction digest is a procedure used in molecular biology to prepare DNA for
analysis or other processing. Restriction digest is most commonly used as part of
the process of the molecular cloning of DNA fragment into a vector. The vector
contains a multiple cloning site where many restriction site may be found, and a
foreign piece of DNA may be inserted into the vector by first cutting the restriction
sites in the vector as well the DNA fragment, followed by ligation of the DNA
fragment into the vector.
Materials required :
1. Ice container

2. Microcentrifuge tubes

3. Purified DNA (>16 ng/µl)

4. Double distilled water (nuclease-free)

5. BSA (4 mg/ml)

6. Restriction enzymes and their respective buffers (10x)

Procedure :

1. About 2 µl of the DNA to be restricted was added to a microcentrifuge tube


placed on ice.

36
2. 2 µl of the restriction enzyme buffer was added.

3. 0.5 µl of BSA was added. (Addition of BSA enhances performance of the


restriction enzyme by providing additional protein to stabilize the enzyme and
balances side-effects arising as a result of enzyme interaction with solid surfaces
like the walls of the tube).

4. 0.5 µl of the restriction enzyme was added.

5. The reaction volume was added to 20 µl with distilled water (The 10x buffer
gets diluted to 1x).

6. The reaction was incubated at 37°C for 30 minutes.

7. To denature the enzymes present in the reaction mix, the mixture was kept at
80°C for 20 minutes and the digested fragments were visualized by agarose gel
electrophoresis.

4.2.2 : Ligation

Principle :

Ligation is the joining of two nucleic acid fragments through the action of an
enzyme. It is an essential laboratory procedure in the molecular cloning of DNA
whereby DNA fragments are joined together to create recombinant DNA
molecules, such as when a foreign DNA fragment is inserted into a plasmid. The
ends of DNA fragments are joined together by the formation of phosphodiester
bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of

37
another. RNA may also be ligated similarly. A co-factor is generally involved in
the reaction, and this is usually ATP or NAD +. Ligation in the laboratory is
normally performed using T4 DNA Ligase enzyme.

Materials required:

1. Linearized plasmid backbones

2. Restriction-digested fragments with complementary overhangs

3. T4 DNA ligase and buffer

4. Double distilled water (nuclease-free)

Procedure:
1. 2 µl (around 25 ng) of the linearized plasmid backbone was added to a
microcentrifuge tube on ice.

2. Equimolar amounts of restricted DNA fragments to was added the


microcentrifuge tube.

3. 1 µl of T4 DNA ligase buffer was added.

4. 0.5 µl of T4 DNA ligase enzyme was added and the volume was made up to 10
µl using double distilled water.

5. The reaction was incubated at 16°C for 30 minutes.

6. The enzyme was heat inactivated at 80°C for 20 minutes.

4.2.3 : Agarose Gel Electrophoresis

38
Principle :

Agarose gel electrophoresis is a method of gel electrophoresis used in molecular


biology to separate a mixed population of macromolecules such as DNA or
proteins in a matrix of agarose.

Materials required:

1. Gel tank apparatus.

2. Agarose. (Low EEO)

3. 10x TBE buffer (108 g TRIS, 55 g boric acid, 40 ml 0.5 M EDTA, Made up to
1l with distilled water)

4. EtBr (10 mg/ml)

5. 6x Gel loading dye

6. DNA ladder

7. Sample DNA

8. UV transilluminator

Procedure :

I. Preparation of agarose gel

1. The agarose was dissolved in 1x TBE buffer to the required concentration (0.8%
is the standard, higher or lower concentrations can be used for separating DNA

39
fragments with very low or very high difference in their lengths respectively).

2. The solution was heated in a microwave to melt the agarose and dissolve it
completely.

3. When it had cooled down to hand-bearable temperature, EtBr was added so that
the final concentration in the gel is about 0.3-0.5 µg/ml.

4. Pour the gel onto a leak-proofed case with a comb inserted and allow the gel to
solidify (The gel turns from completely transparent to cloudy after it solidifies).

II. Loading and running

1. The DNA was mixed with the loading dye (1x) (1:1) on a piece of parafilm
stuck to the work bench.

2. The mixture was loaded into the well in the gel.

3. The DNA ladder was loaded into the well.

4. The gel was run at 60-90 V.

5. The resolved DNA bands were viewed under the UV transilluminator.

4.2.4 : Competent cell preparation

Principle :

Competence is the ability of a cell to take up extrachromosomal DNA. Rapidly


dividing cells can be made competent to take up plasmid DNA by treatment with
CaCl2. CaCl2 interacts with the host cell membrane and makes the membrane more

40
permeable to extracellular plasmid DNA.
Materials required :

1. Escherichia coli cells ( DH5α strain was used)

2. SOB medium (2 % w/v tryptone, 0.5% w/v yeast extract, 8.56mM NaCl, 2.5mM
KCl, 10mM MgCl2, 10mM MgSO4)

3. CCMB80 buffer (pH 6.4) (10mM potassium acetate (pH 7), 80mM CaCl2.2
H2O, 20mM MnCl2.2 H2O, 10mM MgCl2.2 H2O, 10% glycerol)

Procedure :

I. Preparation of seed stocks

1. Single colonies of E.coli were inoculated to 2 ml of SOB medium and shake


overnight.

2. Add glycerol to 15%.

3. Aliquot 1 ml samples and store at -80°C.

II. Preparation of Competent Cells

1. 250 ml of SOB medium was inoculated with 1 ml vial of seed stock and grow at
20°C to an OD of 0.3.

2. The cells were pelleted by centrifuging at 3000xg for 10 minutes at 4°C.

3. The pellet was resuspended in 80 ml of cold CCMB80 buffer.

41
4. Incubation on ice was done for 10 minutes.

5. The contents was centrifuged at 3000xg for 10 minutes at 4°C.

6. The cell pellet was resuspended in 10 ml of ice cold CCMB80 buffer.

7. 50 µl samples were aliquoted and stored at -80°C.

4.2.5 : Transformation :

Principle :

The competent cells take up the DNA provided externally. They then grow
selectively on the plates having the antibiotic towards which they are carrying the
resistance in their newly acquired plasmid DNA.

Materials required :

1. Competent cells

2. Plasmid DNA

3. Ice box

4. Boiling water bath

5. SOC medium (filter-sterilized 20% glucose solution was added to 1l of SOB


medium)

Procedure :

1. The competent cells were thawed on ice for about 15-20 minutes.

42
2. 50 µl of competent cells was pipetted into a 2 ml tube placed on ice.

3. 1 µl of DNA was added to the tube.

4. The contents of the tube was mixed well and incubated on ice for 30 minutes.

5. Heat shock was provided by placing the cells at 42°C for 1 minute and
immediately tube was transferred to ice and incubated for 5 minutes.

6. 200 µl of SOC medium was added to the tube and incubated at 37°C for 2
minutes.

7. Spread plate was done using 20 µl of the culture onto the chloramphenicol -
containing plate.

8. The plate was incubated overnight and the transformation efficiency was
checked.

4.2.6 : Plasmid DNA Isolation :

Materials required:

1. Plasmid DNA - containing cell culture.

2. P1 buffer (pH 8.0, store at 4°C)(50 mM TRIS-Cl pH 8.0, 10 mM EDTA, 100


µg/ml RNaseA)

3. P2 buffer (200 mM NaOH, 1% (w/v) SDS)

4. N3 buffer (pH 4.8) (4.2 M Guanidium hydrochloride, 0.9 M Potassium acetate)

43
5. Isopropanol

6. 70% ethanol

7. 0.5x TE buffer

Procedure :

1. The cells were pelleted by centrifuging them at 4000 rpm for 10 minutes.

2. The pellet was resuspended in 250 µl of P1 buffer and vortexed.

3. 250 µl of P2 buffer was added and mixed by inverting the tube.

4. 350 µl of N3 buffer was added and immediately mixed by inverting the tube
while avoiding localized precipitations.

5. Centrifugation was done for 10 minutes at 13000 rpm.

6. The supernatant was transferred to a fresh tube.

7. Around 500 µl of isopropanol was added.

8. Centrifugation was done at 8000 rpm for 10 minutes.

9. The supernatant was discarded and the pellet was washed twice using 70%
ethanol.

10. The tubes were air dried and the pellet was dissolved in 20 µl of 0.5x TE
buffer. The tubes were stored at -20°C.

44
CHAPTER 5
RESULTS AND DISCUSSION

5.1 : IN SILICO STUDIES


5.1.1 : First tier elimination

The AMPA Antimicrobial Sequence Scanning System (Torrent et al., 2012) was
used to design only a limited number of peptides based on the antimicrobial indices
of the individual amino acids. The lower the antimicrobial index of an amino acid,
the better the candidate for designing an antimicrobial peptide (AMP) (Torrent et
al., 2012). Decapeptides consisting of the arginine, lysine, cysteine, tryptophan and
tyrosine, each, combined with all the other 20 amino acids were written down
along with the reverse peptide for each peptide designed. The total came to 175
decapeptides.

45
Figure 11 : Mean antimicrobial indices of decapeptides consisting of two alternating amino
acid residues.

The window was set as 5 and the threshold as 0.225 and the mean antimicrobial
index was determined for each of the 175 peptides. The peptides were ranked
according to the results, with the peptides with the lowest mean antimicrobial
indices being considered the best. It was observed that the reverse decapeptide for
any decapeptide always had the same mean antimicrobial index, even though,
maximum and the minimum antimicrobial index for every amino acid within the
peptide showed variation between the two peptides. Only those decapeptide with
an index below 0.24 were selected for the next step. Only 80 decapeptides made it
through this process.

46
5.1.2 : Second tier elimination

In the APD3 Antimicrobial Peptide Database website, the Peptide design ->
Improve Peptide options were selected and each of the 80 peptides were entered as
query. Based on the above observation, all the decapeptides that did not contain
any hydrophobic residues were eliminated. Only 49 decapeptides remained, in
which 32 had a predicted net positive charge, a property viewed as favorable while
designing an AMP, while 17 had predicted no net charge. Nevertheless, the
physical and chemical properties for all 49 peptides were determined using
ExPASy ProtParam website. The Instability Index varied from a highest score of
295.4 for the peptide N – RWRWRWRWRW – C to a lowest score of -67.41 for
the peptides N – KIKIKIKIKI – C, N – IKIKIKIKIK – C, N – KLKLKLKLKL –
C, N – LKLKLKLKLK – C, while for approximately 1/3 of the peptides, it was a
constant value of 9. The lower the Instability Index, the better. The Theoretical pI
varied from a highest value of 12.6 for about 1/3 of the peptides to a lowest value
of 5.49 for N – CYCYCYCYCY – C and N – YCYCYCYCYC – C alone.

5.1.3 : Third tier elimination

The CAMP website AMP prediction tool was used.All 4 outputs were considered
and only the peptides which scored a maximum positive result in at least 3 output
formats were selected. 14 peptides were selected out of this process. Among these,
11 had a predicted net positive charge while 3 had predicted no net charge.

47
5.1.4 : Is it a signal peptide?
Whether the designed peptides could be signal peptides or could contain a signal
peptide or not was predicted using the SignalP 4.1 Server. It was observed that
only one peptide N – IWIWIWIWIW – C was predicted to be a signal peptide,
while for all the other peptides, the prediction turned out to be negative. Even for
this peptide the prediction was positive only when the host was selected to be
eukaryotic.

Figure 12a

48
Figure 12b
Figure 12: SignalP 4.1 Results:

They show the C,S and Y scores as obtained for the peptide N – IWIWIWIWIW – C from the
SignalP 4.1 server. The output was Name=IWIWIWIWIW_01 SP='YES' Cleavage site between
pos. 2 and 3: W-IW D=0.460 D-cutoff=0.450 Networks=SignalP-noTM

5.1.5 : Anticancer properties


To test whether the 14 decapeptides had any anticancer properties, the AntiCP
website was used.(Tyagi et al., 2013) The SVM Score which shows a direct
correlation to the peptide’s anticancer properties(Tyagi et al., 2013), showed a
maximum score of 0.99 only for N – KLKLKLKLKL – C and N –
LKLKLKLKLK – C.
The final 14 predicted antimicrobial peptides along with their different properties
have been given in tables 2 and 3.
5.1.6 : Secondary structure prediction
The secondary structures of the 14 peptides were determined using PEP-FOLD 3
and the output was fed into Vega ZZ Molecular Modelling software. The cluster
calculation was performed on all the peptides using LIQUID POPC as solvent,

49
thickness = 20Å, overlap = 0.8Å, type = layer and cluster position as geometry
center. The results are given from Figures 13 to 26.

Table 2 : The comprehensive table for the 14 decapeptides


(1) indicates that all cysteines have been converted to cystines and (2) means that all cysteines
have been reduced. N- WRWRWRWRWR - C has been abbreviated as WR.
S Pepti AMPA
no des AI APD3 ExPASy ProtParam
Estimated Half Life
WWH BI MR E.coli
(kcal/ (kcal/ Theoretica EC in yeast in GRA Instability
Mean mol) mol) l pI (M-1 cm-1) vitro in vivo vivo VY Index
1 RC 0.183 2.85 6.82 9.6 - 1hr 2min 2min -1 9
250(1),
2 KC 0.185 3.75 2.13 9.26 0(2) 1.3hr 3min 3min -0.7 9
3 WR 0.187 -5.2 6.29 12.6 27500 2.8hr 3min 2min -2.7 238.12
4 RW 0.187 -5.2 6.29 12.6 27500 1hr 2min 2min -2.7 295.4
5 KW 0.189 -4.3 1.6 10.6 27500 1.3hr 3min 3min -2.4 9
6 WK 0.189 -4.3 1.6 10.6 27500 2.8hr 3min 2min -2.4 9
7 IK 0.204 3.4 0.31 10.6 - 20hr 30min >10hr 0.3 -67.41
8 KF 0.23 -0.7 1.28 10.6 - 1.3hr 3min 3min -0.55 -51.12
9 FK 0.23 -0.7 1.28 10.6 - 1.1hr 3min 2min -0.55 -66.15
10 KL 0.23 2.15 0.31 10.6 - 1.3hr 3min 3min -0.05 -67.41
11 LK 0.23 2.15 0.31 10.6 - 5.5hr 3min 2min -0.05 -67.41
The peptides listed above have a net positive charge and the peptides listed below have net 0
charge

50
625(1),
12 CC 0.215 -2.4 -1.27 5.46 0(2) 1.2hr >20hr >10hr 2.5 9
13 WW 0.223 -18.5 -2.32 5.52 55000 2.8hr 3min 2min -0.9 9
14 IW 0.237 -10.8 -3.62 5.52 27500 20hr 30min >10hr 1.8 9

Table 3 : The comprehensive table for the 14 decapeptides


SVM - Support Vector Machine, RFC - Random Forest Classifier, ANN - Artificial Neural
Network, DAC - Discriminant Analysis Classifier
Pepti SignalP
S no des CAMP 4.1 AntiCP
Signal
SVM RFC ANN DAC Peptide? SVM Score
0.6165
1 RC 1 (AMP) (AMP) NAMP 0.996 (AMP) NO 0.45 (AntiCP)
0.518 0.602
2 KC (AMP) (AMP) AMP 0.98 (AMP) NO 0.46 (AntiCP)
0.726
3 WR 1 (AMP) (AMP) NAMP 1 (AMP) NO 0.45 (AntiCP)
0.7215
4 RW 1 (AMP) (AMP) NAMP 0.999 (AMP) NO 0.45 (AntiCP)
0.7725
5 KW 1 (AMP) (AMP) AMP 1 (AMP) NO 0.48 (AntiCP)
0.778
6 WK 1 (AMP) (AMP) AMP 1 (AMP) NO 0.48 (AntiCP)

51
0.438
7 IK (NAMP) 0.8 (AMP) AMP 0.996 (AMP) NO 0.33 (AntiCP)
0.635
8 KF 1 (AMP) (AMP) AMP 0.953 (AMP) NO 0.47 (AntiCP)
0.6645
9 FK 1 (AMP) (AMP) AMP 0.977 (AMP) NO 0.47 (AntiCP)
0.669
10 KL 0.986 (AMP) (AMP) AMP 0.99 (AMP) NO 0.99 (AntiCP)
0.6985
11 LK 0.991 (AMP) (AMP) AMP 0.994 (AMP) NO 0.99 (AntiCP)
The peptides listed above have a net positive charge and the peptides listed below have net 0
charge
0.6915
12 CC 1 (AMP) (AMP) AMP 0.956 (AMP) NO 0.36 (AntiCP)
0.5616
13 WW 1 (AMP) (AMP) AMP 1 (AMP) NO 0.36 (AntiCP)
0.5795
14 IW 0.91 (AMP) (AMP) AMP 0.999 (AMP) YES 0.4 (AntiCP)

52
Figure 13 : N-RCRCRCRCRC-C

The peptide’s secondary structure as predicted using PEP-FOLD (left) and N-RCRCRCRCR -C
peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right).

Figure 14 : N-KCKCKCKCKC-C

The peptide’s secondary structure as predicted using PEP-FOLD (left) and N-KCKCKCKCKC-
C peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

53
Figure 15 : N-WRWRWRWRWR-C

The peptide’s secondary structure as predicted using PEP-FOLD (left) and N-


WRWRWRWRWR-C peptide visualized in a liquid POPC cluster. The blue molecules are water
molecules and the white molecules are POPC molecules.(right)

Figure 16 : N-RWRWRWRWRW-C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-RWRWRWRWRW-


C peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right).

54
Figure 17 : N-KWKWKWKWKW–C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-KWKWKWKWKW-


C peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

Figure 18 : N-WKWKWKWKWK-C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-WKWKWKWKWK-


C peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

55
Figure 19 : N-IKIKIKIKIK-C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-IKIKIKIKIK-C


peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

Figure 20 : N-KFKFKFKFKF–C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-KFKFKFKFKF-C


peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

56
Figure 21 : N-FKFKFKFKFK–C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-FKFKFKFKFK-C


peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

Figure 22 : N-KLKLKLKLKL–C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-KLKLKLKLKL-C


peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right).

57
Figure 23 : N-LKLKLKLKLK–C

The peptide’s secondary as predicted by PEP-FOLD (left) and N-LKLKLKLKLK-C peptide


visualized in a liquid POPC cluster. The blue molecules are water molecules and the white
molecules are POPC molecules.(right)

Figure 24 : N-CCCCCCCCCC–C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N-CCCCCCCCCC-C


peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

58
Figure 25 : N-WWWWWWWWWW-C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N -


WWWWWWWWWW - C peptide visualized in a liquid POPC cluster. The blue molecules are
water molecules and the white molecules are POPC molecules.(right).

Figure 26 : N-IWIWIWIWIW-C

The peptide’s secondary structure as predicted by PEP-FOLD (left) and N - IWIWIWIWIW - C


peptide visualized in a liquid POPC cluster. The blue molecules are water molecules and the
white molecules are POPC molecules.(right)

59
As it can be seen from the above figures, all of the top 14 peptides become
completely surrounded by POPC molecules during clustering. This is a favorable
characteristic for an antimicrobial peptide as it is observed that the most effective
antimicrobial peptides are those that can get inserted into the phospholipid lipid
bilayer (Wenzel et al., 2014).

5.1.7 : Selection of antimicrobial peptide for laboratory work

Out of the 14 peptides, the one having arginine and tryptophan was selected for
proceeding to lab work based on previous evidence of its antimicrobial
activity(Wenzel et at., 2014).

60
Table 4 : Comparison of random peptides having arginine and tryptophan

5.1.8 : Plasmid design

The backbone was 2070 bp, the lac cassette is 1633 bp and the AMP sequence ( N
- MWRWRWR - C ) is 21bp. The total size of the cassette was 3724bp.

61
Figure 27 : Plasmid design for the lac - AMP cassette.

5.2 : LABORATORY WORK

5.2.1 : Plasmid construction

The plasmid was constructed using the standard molecular biology techniques such
as restriction, ligation and polymerase chain reaction. The backbone pSB1C3 with
the lac promoter was cut using SpeI and PstI. The AMP sequence was separately
cut using XbaI and PstI. The ligation was done using T4 DNA ligase. An
extremely faint band was observed at around 4kb. This corresponds to the actual

62
size of the antimicrobial peptide of 3724 kb.

Figure 28: The antimicrobial peptide cassette viewed in a gel.

5.3 : DISCUSSION

This project was done to see whether really small peptides can act as antimicrobial
peptides. There appear to be some characteristics that appear to be essential for a
peptide to display antimicrobial properties. These include the peptide length,
amino acid composition, hydrophobicity of the amino acids(Wenzel et al., 2014),
the peptide secondary structure, the overall charge of the peptide and whether the
peptide is a signal peptide. Antimicrobial peptides usually have a large proportion
of hydrophobic residues. Positively charged amino acids like arginine and lysine
are also favored to be a part of antimicrobial peptides.(Wenzel et al., 2014) The

63
antimicrobial activity also increases as the peptide loses all its non antimicrobial
regions i.e. as the peptide becomes smaller. It is seen that AMPs are usually one of
the 4 classes i) α helical, ii) β stranded due to the presence of 2 or more disulfide
bonds, iii) β-hairpin or loop due to the presence of a single disulfide bond and/or
cyclization of the peptide chain, and iv) extended. Sometimes they can also have
an undefined structure.

It is observed that among the 14 best peptides predicted to have a good


antimicrobial activity in this study, the only ones with the α helical conformation
are N-RWRWRWRWRW-C, N-WRWRWRWRWR-C, N-WWWWWWWWW-
C, N-IWIWIWIWIW-C. Though the peptide N-CCCCCCCCCC-C does not have
any hydrophobic residues, it still shows good promise, since it passed all three
levels of AMP prediction.

The cluster calculation shows that all the 14 peptides show insertion into the
Liquid POPC solvent. POPC is used to as a substitute for phospholipid bilayer. The
results observed are consistent with the fact that many good antimicrobial peptides
show insertion into the phospholipid bilayer(Wenzel et al., 2014).

For testing the AMP in the lab, the sequence N-MWRWRWR-C was selected
because 1. It showed promise as an antimicrobial peptide during the study and 2.
There was experimental evidence that a hexapeptide having alternating arginine
and tryptophan has good antimicrobial activity.

The ligated AMP cassette was successfully created in the laboratory, and the result
is shown in Figure 28.

64
CHAPTER 6
CONCLUSION

175 decapeptides were proposed and 14 peptides were finalized as good


antimicrobial candidates based on antimicrobial index, instability index,
hydrophobicity and other properties. The finalized peptides were modelled and
cluster calculation was performed on all the peptides using Liquid POPC as
solvent. A plasmid construct was designed using only arginine and tryptophan for
the antimicrobial peptide and the plasmid was constructed in the laboratory.
Arginine and tryptophan were used because
1. They have relatively low antimicrobial index (Torrent et al., 2011)
2. The present study proved that arginine and tryptophan can be a part of an
effective antimicrobial peptide.
3. A hexapeptide having only areginine and tryptophan was proved to have an
excellent antimicrobial effect.(Wenzel et al., 2014)
It was then transformed into E.coli and the band visualized in an agarose gel using
electrophoresis. In the future, the 14 peptides have to aligned with well known
AMPs and their physical and chemical properties should also be compared.

65
CHAPTER 7
REFERENCES

1. Ausubel, F.M (1999); ‘Short Protocols in Molecular Biology’; John Wiley &
Sons, Ed. 4, pp.A1-A36.

2. Brogden Kim. (2005); ‘ANTIMICROBIAL PEPTIDES: PORE FORMERS OR


METABOLIC INHIBITORS IN BACTERIA?’; Nature Reviews Microbiology,
Vol.3, No.3, pp.238-250.

3. Dürr, U.H.N., Sudheendra, U.S., Ramamoorthy, A. (2006); "LL-37, the only


human member of the cathelicidin family of antimicrobial peptides"; Biochimica
et Biophysica Acta (BBA) - Biomembranes, Vol.1758, No.9, pp.1408–1425.

4. Giuliani, A∗ ., Pirri, G., Nicoletto, F.S (2007); ‘Antimicrobial peptides: an


overview of a promising class of therapeutics’; VERISTA Central European
Journal of Biology, Vol.2, No.1, pp.1 – 33.

5. Hancock, R *., Rozek, A (2002); ‘Role of membranes in the activities of


antimicrobial cationic peptides’; FEMS Microbiology Letters, Vo..206, No.2,
pp.143-149.

6. Hancock, R., Sahl, H.S (2006); ‘Antimicrobial and host-defense peptides as new
anti-infective therapeutic strategies’; Nature Biotechnology, Vol.24, No.12,
pp.1551-1557.

7. Lamiable, A., Thévenet, P., Rey, J., Vavrusa, M., Derreumaux, P., Tufféry, P.
(2016); ‘PEP-FOLD3: faster de novo structure prediction for linear peptides in
solution and in complex.’; Nucleic Acids Research, Vol.44, No.1, pp.449-454.

8. Matsuzaki, K (2008), ‘Control of cell selectivity of antimicrobial peptides’;


Biochimica et Biophysica Acta (BBA), Vol.1788, No.8, pp.1687-1692.

66
9. Mátyus, E., Kandt, C., Tieleman, DP (2007); ‘Computer Simulation of
Antimicrobial Peptides’ ; Current Medicinal Chemistry, Vol.14, No.26, pp.2789-
2798.

10. Wang, G., Li ,X., Wang ,Z (2016); ‘APD3: the antimicrobial peptide database
as a tool for research and education’; Nucleic Acid Research, Vol.44, No.1,
pp.D1087–D1093.

11. Petersen, N.T., Brunak, S., Heijne, vG., Nielsen, H (2011); ‘SignalP 4.0:
discriminating signal peptides from transmembrane regions’; Nature
Methods, Vol.8, No.10, pp.785-786.

12. Reddy, K.V., Yedery, R.D., Aranha, C (2004). "Antimicrobial peptides:


premises and promises". International Journal of Antimicrobial Agents. Vol.24,
No.6, pp.536–547.

13. Sitaram, N., Nagaraj, R., (2002); "Host-defense antimicrobial peptides:


importance of structure for activity"; Current Pharmaceutical Design, Vol.8, No.9,
pp.727–742.

14. Torrent, M., Di Tommaso, P., Pulido, D., Nogués, MV, Notredame, C., Boix,
E., Andreu, D (2011); ‘AMPA: an automated web server for prediction of protein
antimicrobial regions’; Bioinformatics, Vol.28, No.1, pp.130-131.

15. Torrent, M1., Nogués, VM., Boix, E (2009 Nov 11); ‘A theoretical approach to
spot active regions in antimicrobial proteins’ , Vol.10, pp.373.

16. Tobias, J.W., Shrader, T.E., Rocap, G., Varshavsky, A (2013); ‘The N-End
Rule in Bacteria’; Science, Vol.254, No.5036, pp.1374 - 1991.

17. Tyagi, A., Kapoor, P., Kumar, R., Chaudhary, K., Gautam, A., Raghava, G.P
(2013) ; ‘In Silico Models for Designing and Discovering Novel Anticancer
Peptides’, Scientific Reports, Vol.3, No.2984, pp.1-8.

67
18. Waghu, F.H., Barai, R.S., Gurun,g P., Idicula, T.S (2015); ‘CAMPR3: a
database on sequences, structures and signatures of antimicrobial peptides.’,
Nucleic Acids Research, Vol.44, No.1, pp.gkv1051v1-gkv1051.

19. Wenzel, M., Chiriac, AI., Otto, A., Zweytick, D., May, C., Schumacher, C.,
Gust, R., Albada, HB., Penkova, M., Krämer, U., Erdmann, R., Metzler-Nolte, N.,
Straus, S.K., Bremer, E., Becher, D., Brötz-Oesterhelt, H., Sahl, H.G., Bandow,
J.E. (2014); ‘Small cationic antimicrobial peptides delocalize peripheral membrane
proteins.’ ; PNAS, Vol.111, No.14, pp. E1409–E1418.

20. Wildman, H., Katherine, A., Lee, D.K., Ramamoorthy, A.* (2003);
‘Mechanism of Lipid Bilayer Disruption by the Human Antimicrobial Peptide, LL-
37†’; Biochemistry ; Vol.42, No.21, pp.6545 – 6558.

21. Yeaman, M.,Yount, N (2003); ‘Mechanism of Antimicrobial Peptide Action


and Resistance’; The American Society for Pharmacology and Experimental
Therapeutics, Pharmacological Reviews, Vol.55, pp.27–55.

68
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