Escolar Documentos
Profissional Documentos
Cultura Documentos
University Of Mumbai
(By Papers)
By
Department of Biotechnology
Matunga , Mumbai.
In the realization of ones goal, man is not an independent identity.It is the encouargement
and guidance of his peer, associates and colleagues that follows him on the road to
success.Keeping this in mind I would like to take the oppurtunity to show my appreciation to
all those who have contributed towards my project, in one way or the other, big or small.
I avail this opportunity to express my deep sense of gratitude to head of radiation medicine
center , BARC, Mumbai ; Dr. M.G.R. Rajan for giving me this opportunity and permission to
commence my project at this esteemed institution and utilize all the required facilities without
reservation.
I express my sincere thanks to my project guide, Dr. (Mrs.) Savita Kulkani. Scientific Officer
G. who monitored my work and subjected to the scrupulous precision. This ensures
successful completion of the project within proviso. Her enthusiasm and love for work have
greatly motivated me. Her contribution to in valuable guidance and stimulating suggestions. I
am very thankful to her for devoting time, guide me to critically evaluate the dissertation to
make it comprehensible and scientifically correct.
I am very thankful to Shri. Muktikant ray, Shri. Kumarswami, Mr. Avik Chakarwarty, Ms.
Megha Sodani and Mr. Pramodkumar Gupta for providing useful insights, help and guidance
during my training period. I have been greatly benefited from their guidance to strengthen
working skill in the laboratory.
I would like to thank Mr. Shaikh for providing me all the necessary requirement without
which poject would not be complete.
I convey special thanks to the Principal, Dr. Suhas Pednekar and Miss. Supriya
Kale,Head of Department of Biotechnology, Ramnarain Ruia College of Arts & Science,
who have given me the golden opportunity to develop this project.
I also specially thank Ms. Rupali Pathanka and all other teachers of Department of
Biotechnology of Ruia College and my co-trainees Rucha, Anushree, Riya, Yashwi for
their support,help and co-operation.
Above all, I thank family for their unwavering inspiration, love and moral support.
Finally, I would like to thank each and every individual who help in making this project a
great learning experience.
(Asmita A. Jogi)
ABSTACT
Tuberculosis bestows some of the major challenges in diagnosis and treatment. The
major challenge is to find the specific mycobacterial antigens that can provide desired
immunoprophylactic response against infection. Additionally, such antigens are required in
larger amounts for carrying out immunological characterization , but are difficult to getting
larger quantities due to slow growth, inherent pathogenicity and difficulty in biomedical
purification from mycobacterial tuberculosis cell. In such situation, overexpression of such an
antigen after cloning in appropriate vector system by recombinant DNA technology seems to
be an ultimate solution.
ESAT 6 being secretory protein is gradually released during the growth of the bacilli
increasing during the culture period. The protein is then translocate across cytoplasmic
membrane and localized in outer cell wall region to ESAT 6 antigen used in gamma interferon
assay to quantify INF-gamma released by T-cells in response to stimulation by mycobacterium
tuberculosis antigens.
Tuberculosis is considered as one of the major health problems throughout the world . Nearly
80% of the tuberculosis cases occur in developing countries. Therefore, prompt diagnosis of
patients with tuberculosis is vital for reducing the incidence of this disease.
ESAT-6 andCFP-10 have been described as dominant antigens recognized by T-cells and
consider as virulence factors in M. tuberculosis These two major proteins facilitate
translocation of M. tuberculosisfrom the phagosome into the host cell cytoplasm at later
stages of infection . There are dilemmas in precise, rapid, and cost-effective diagnostic tools
of tuberculosis, therefore evaluation of M. tuberculosisspecific antigens such as ESAT-6
andCFP-10 to achieve targets for accurate diagnosis is recommended. The aim of this study
was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M.
tuberculosis in soluble form for future diagnostic purposes by Interferon- Release Assays
(IGRAs) using the ESAT-6/CFP-10 antigens.
REVIEW OF LITERATURE
Tuberculosis(TB) is the leading cause of death in the world from a bacterial infectious
disease. The disease affects 1.8 billion people/year which is equal to one-third of the entire
world population.
In the United States TB is on the decline. In 2007 a total of 13,293 cases were
reported. The TB rate declined to 4.4 cases per 100,000 population, the lowest recorded rate
since national reporting began in 1953. Despite this overall improvement, progress toward
TB elimination has slowed in recent years; the average annual percentage decline in the TB
rate slowed from 7.3% per year during 1993--2000 to 3.8% during 2000--2007. Also, since
1993 there has been a gradual decline in the number of TB patients with co-infection with
HIV, and the number of cases of multiple drug-resistant TB has gradually dropped.
On the other hand, the proportion of TB cases contributed by foreign-born persons has
increased each year since 1993. In 2007 the TB rate in foreign-born persons in the United
States was 9.7 times higher than in U.S.-born persons. In many states, especially in the West,
the upper Midwest, and the Northeast, most new cases of TB now occur in individuals who
are foreign born.
General Characteristics
Detoxification of oxygen radicals. MTB interferes with the toxic effects of reactive oxygen
intermediates produced in the process of phagocytosis by three mechanisms:
1. Compounds including glycolipids, sulfatides and LAM down regulate the oxidative
cytotoxic mechanism.
2. Macrophage uptake via complement receptors may bypass the activation of a respiratory
burst.
3. The oxidative burst may be counteracted by production of catalase and superoxide
dismutase enzymes.
Antigen 85 complex. This complex is composed of a group of proteins secreted by MTB that
are known to bind fibronectin. These proteins may aid in walling off the bacteria from the
immune system and may facilitate tubercle formation.
Slow generation time. Because of MTB's slow generation time, the immune system may not
readily recognize the bacteria or may not be triggered sufficiently to eliminate them. Many
other chronic disease are caused by bacteria with slow generation times, for example, slow-
growing M. leprae causes leprosy,Treponema pallidum causes syphilis, and Borrelia
burgdorferi causes Lyme disease.
High lipid concentration in cell wall. This accounts for impermeability and resistance to
antimicrobial agents, resistance to killing by acidic and alkaline compounds in both the
intracellular and extracellular environment, and resistance to osmotic lysis via complement
deposition and attack by lysozyme
Cord factor. Cord factor (trehalose 6, 6' dimycolate) is a glycolipid found in the cell walls of
mycobacteria, which causes the cells to grow in serpentine cords. It is primarily associated
with virulent strains of MTB. It is known to be toxic to mammalian cells and to be an
inhibitor of PMN migration. Its exact role in MTB virulence is unclear, although it has been
shown to induce granulomatous reactions identical to those seen in TB.
M. tuberculosis virulence is studied both in tissue culture, using macrophages, dendritic cells
or pneumocytes, and in animal models, primarily mice. Tissue culture models are easier and
more humane to work with and give faster results, but they are limited to studying early
stages of infection. Ultimately, only in animal models can all the stages of TB be studied.
Since sequencing the Mycobacterium tuberculosis genome in 1998, genetic methods are more
commonly used to study the bacterium's virulence. The usual genetic approaches to study
virulence are to disrupt, inactivate, modify, delete or complement a gene and assess the
effects in the macrophage or mouse model.
Of approximately 4,000 genes in the Mycobacterium tuberculosis genome, 525 are involved
in cell wall and "cell processes", 188 genes encode regulatory proteins, and 91 genes are
involved in "virulence, detoxification and adaptation". Over 200 genes are identified as
encoding enzymes for the metabolism of fatty acids. This large number of M.
tuberculosis enzymes that putatively use fatty acids may be related to the ability of the
pathogen to grow in the tissues of the infected host, where fatty acids may be the major
carbon source. This is thought to be an important aspect of M. tuberculosis physiology during
infection. The point is that these genes, as well as those from other classes, may be directly or
indirectly involved in virulence.
Some of these genes and their products or activities are described below :
19-kDa protein. The 19-kDa protein is a secreted antigenic protein that is immunologically
recognized by T cells and sera from TB patients. When M. tuberculosis enters macrophages
and other phagocytic cells, this surface-exposed glyco-lipoprotein is thought to cause host
signaling events as it interacts with its receptor, TLR2. There is suggestive but inconclusive
evidence that mutant strains of Mycobacterium tuberculosis deficient in production of the 19-
kDa protein are rapidly cleared from the lungs and spleen of infected mice. Adding back the
wild-type M. tuberculosis gene to these strains allowed growth in lungs that was similar to
the growth of standard wild-type strains, which suggested that the 19-kDa protein is essential
for virulence. It has been reported that addition of the purified 19-kDa protein to human
macrophages causes upregulation of the Th1 cytokine IL-12. Similarly, the 19-kDa protein
can active human neutrophils .
Glutamine synthase. The enzyme is not secreted into culture filtrates during growth but
results from cell leakage and lysis. glnA1 mutations have not been made in M. tuberculosis,
but the specific glutamine synthase inhibitor, L-methionine-SR-sulfoximine (MSO), inhibits
the growth of Mycobacterium tuberculosis in vitro and in macrophages but has no effect on
nonpathogenic mycobacteria. In addition to its essential role in nitrogen metabolism,
glutamine synthase is involved in the synthesis of a poly-L-glutamate-glutamine cell wall
component found in pathogenic mycobacteria. These findings have led to the suggestion that
this enzyme is a possible target for the development of new drugs which have less toxicity
than MSO for humans.
Mas. Mas encodes mycocerosic aid synthase, an enzyme that catalyzes the synthesis of long-
chain, multiply methylated branched fatty acids, called mycocerosic acids, that are found
only in pathogenic mycobacteria.
FbpA. Mycobacteria have three mycolyl-transferase enzymes, encoded by three genes, fbpA,
fbpB, and fbpC, that transfer long-chain mycolic acids to trehalose derivatives. The proteins
can also bind the cell matrix protein fibronectin. The Fbp proteins are also found in the
culture filtrate and are known as the antigen 85A, 85B, and 85C complex (Antigen 85
complex). The three fbp genes have been separately inactivated, but only the M.
tuberculosis fbpA mutant showed severely attenuated growth in human and murine
macrophages. The observation that these proteins produce a formidable immunologic
response led to the creation of a new live vaccine that was made by introducing the M.
tuberculosis fbpB gene into M. bovis BCG. This recombinant strain shows better protection
against virulent M. tuberculosis infection than does the parent BCG strain in a guinea pig
model.
OmpA. OmpA is a porin-like protein that can form pores in liposomes, a general property of
porin family proteins. ompA expression is induced by low pH, as well as by engulfment into
macrophages. An ompA mutant demonstrated the following phenotypes: although it showed
delayed growth at acid pH, it ultimately grew at wild-type levels; it could not take up small
molecules like serine at low pH; it showed reduced ability to grow in both human and murine
macrophages. This suggested that the environment encountered by M. tuberculosis during
infection is acidic, and that OmpA played a role in the bacterial response to this condition.
When the mbtB gene of M. tuberculosis is inactivated the resulting mutant strain is
unable to synthesize the two mycobactin-derived siderophores. The mutant showed wild-type
growth in iron-rich media, but grew poorly when iron is limited. It also grew more slowly
than the wild type in human macrophages indicating that the phagosome containing
mycobacteria may be low in iron. Furthermore, excess iron exacerbates the progression of TB
in humans and animal models. These observations suggest that iron availability, made
possible through the activities of siderophores, promotes the growth and virulence of the M.
tuberculosis.
Oxidative stress proteins. Most aerobic organisms have enzymes that degrade peroxides and
superoxide, which are normal byproducts of aerobic respiration, but also are toxic oxygen
radicals. These enzymes, generally superoxide dismutases, catalases and peroxidases, are also
important for the response to various external oxidative stresses. Since phagocytic cells
produce oxygen radicals during the respiratory burst to kill invading bacteria, it is not
surprising that these enzymes may contribute to M. tuberculosis virulence. Enzymes found
inM. tuberculosis that combat oxygen radicals include AhpC, an alkyl hydroperoxide
reductase that detoxifies organic hydroxyperoxides, and SodA and SodC, two species of
superoxide dismutase that degrade superoxides, which are normal by-products of aerobic
respiration and are also produced by the phagocytic respiratory burst.
Nitrate reductase. . M. tuberculosis was originally thought to be an obligate aerobe, but
there are numerous experimental indicators that the bacterium can grow in microareophilic
environments, especially during the later stages of infection, e.g., in lung granulomas. Wild
type M. tuberculosis has been shown to possess an inducible nitrate reductase (NarG encoded
by narG) which allows respiration using NO3 as a final electron acceptor. If anaerobic or
microareophilic growth is an important feature of M. tuberculosis physiology during
infection, the existence of nitrate reductase could be a significant factor in sustaining growth
under these conditions
Adherence
The diagnosis of tuberculosis requires detection of acid-fast bacilli in sputum via the
Ziehl-Neelsen method as previously described. The organisms must then be cultured from
sputum. First, the sputum sample is treated with NaOH. This kills other contaminating
bacteria but does not kill the MTB present because cells are resistant to alkaline compounds
by virtue of their lipid layer. Skin Testing is performed as the tuberculin or Mantoux
test. PPD (purified protein derivative) is employed as the test antigen in the Mantoux test.
PPD is generated by boiling a culture of MTB, specifically Old Tuberculin (OT). 5 TU
(tuberculin units), which equals 0.000lmg of PPD, in a 0.1 ml volume is intracutaneously
injected in the forearm. The test is read within 48-72 hours.
The test is considered positive if the diameter of the resulting lesion is 10 mm or greater. The
lesion is characterized by erythema (redness) and swelling and induration (raised and hard).
90% of people that have a lesion of 10 mm or greater are currently infected with MTB or
have been previously exposed to MTB. 100% of people that have a lesion of 15 mm or
greater are currently infected with MTB or have been previously exposed to MTB. False
positive tests usually manifest themselves as lesser reactions. These lesser reactions could
indicate prior exposure or infection with other mycobacteria or vaccination with BCG.
However, in places were the vaccine is not used, lesser reactions should be regarded as highly
suspicious.
False negatives are more rare than false positives but are especially common in AIDS
patients as they have an impaired CMI response. Other conditions such as malnutrition,
steroids, etc., can rarely result in a false negative reaction.
Tuberculosis Treatment
Hence, tuberculosis is usually treated with four different antimicrobial agents The
course of drug therapy usually lasts from 6-9 months. The most commonly used drugs are
rifampin (RIF) isoniazid (INH), pyrazinamide (PZA ) and ethambutol (EMB) or streptomycin
(SM). When adherence with the regimen is assured, this four-drug regimen is highly
effective. Based on the prevalence and characteristics of drug-resistant organisms, at least
95% of patients will receive an adequate regimen (at least two drugs to which their organisms
are susceptible) if this four-drug regimen is used at the beginning of therapy. Furthermore, a
patient who is treated with the four-drug regimen, but who defaults therapy, is more likely to
be cured and not relapse when compared with a patient treated for the same length of time
with a three-drug regimen.
ESAT-6
ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M.
tuberculosis) is an important virulence factor, inactivation of which leads to reduced
virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10
(ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed
mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex
does not suggest presence of enzymatic or DNA-binding activities. Therefore, we
hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could
be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening,
we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (2M), which
was further confirmed by other assays, like GST pull down, co-immunoprecipitation and
surface plasmon resonance. The C-terminal six amino acid residues (9095) of ESAT-6 were
found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts
with 2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum
where it sequesters 2M to inhibit cell surface expression of MHC-I-2M complexes,
resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-
6:2M complex could be detected in pleural biopsies of individuals suffering from pleural
tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine
the host adaptive immune responses to establish a successful infection. Identification of such
novel interactions may help us in designing small molecule inhibitors as well as effective
vaccine design against tuberculosis.
Any E. coli host strain containing both the expression (pQE) and the repressor
(pREP4) plasmids can be used for the production of recombinant proteins. The QIAexpress
System uses E. coli strain M15[pREP4] which permits high-level expression and is easy to
handle. E. coli strains that harbor the lacIq mutation, produce enough lac repressor to
efficiently block transcription, and are ideal for storing and propagating pQE plasmids. These
strains can also be used as expression hosts for expressing nontoxic proteins, but they may be
less efficient than the M15[pREP4] strain, and expression is regulated less tightly than in
strains harboring the pREP4 plasmid. If the expressed protein is toxic to the cell, leaky
expression before induction may result in poor culture growth or in the selection of deletion
mutants which grow faster than bacteria containing the correct plasmid. Note that E. coli
strains M15 do not harbour a chromosomal copy of the lacIq mutation, so pREP4 must be
maintained by selection for kanamycin resistance
pQE vectors
Any E. coli host strain containing both the expression (pQE) and the repressor
(pREP4)plasmids can be used for the production of recombinant proteins. The QIAexpress
Systemuses E. coli strain M15[pREP4] which permits high-level expression and is easy to
handle.. The M15 strain derived from E. coli K12 and have the phenotype NaIS, StrS, RifS,
Thi, Lac, Ara+, Gal+, Mtl, F, RecA+, Uvr+, Lon+. E. coli strains that harbor the lacIq
mutation, such as XL1 Blue, JM109 and TG1, produce enough lac repressor to efficiently
block transcription, and are ideal for storing and propagating pQE plasmids. These strains can
also be used as expression hosts for expressing nontoxic proteins, but they may be less
efficient than the M15[pREP4] strain, and expression is regulated less tightly than in strains
harboring the pREP4 plasmid. If the expressed protein is toxic to the cell, leaky expression
before induction may result in poor culture growth or in the selection of deletion mutants
which grow faster than bacteria containing the correct plasmid. Note that E. coli strain M15
do not harbour a chromosomal copy of the lacIq mutation, so pREP4 must be maintained by
selection for kanamycin resistance.
PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR
methods typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp),
although some techniques allow for amplification of fragments up to 40 kbp in size. The
amount of amplified product is determined by the available substrates in the reaction, which
become limiting as the reaction progresses.
A basic PCR set up requires several components and reagents. These components include:
The PCR is commonly carried out in a reaction volume of 10200 l in small reaction tubes
(0.20.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction
tubes to achieve the temperatures required at each step of the reaction . Many modern thermal
cyclers make use of the Peltier effect, which permits both heating and cooling of the block
holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes
permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal
cyclers have heated lids to prevent condensation at the top of the reaction tube. Older
thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a
ball of wax inside the tube.
Design of the oligonucleotide primer being the most important factor that influence
the efficiency and specificity of the amplification reaction, careful designing of primers is
required to obtain the desired products in high yield, to suppress amplification of unwanted
sequences and to facilitate subsequent manipulation of the amplified product. Since the
primers so heavily influence the success or failure of PCR protocols, it is ironic that the
guidelines for their design are largely qualitative and are based more on common sense than
on well understood thermodynamic or structural principles.
3. Deoxynucleoside triphosphates(dNTPs)
Standard PCRs contain equimolar amounts of all four dNTPs. Concentrations of 200-
250M of each dNTP recommended for Taq polymerase in reactions containing1.5 mM
MgCl2. In a 50 uL reaction, these amounts should allow synthesis of ~6-6.5g DNA which
should be sufficient even for multiplex reaction in which eight or more primer pairs are used
at the same time. High concentrations of dNTPs (>4mM) are inhibitory, perhaps because of
sequestering of Mg2+. However, a satisfactory amount of amplified product can be produced
with dNTP concentrations as low as 20M- 0.5-1.0pM of an amplified fragment ~1 kb in
length. To avoid problems, stocks of dNTPs should be stored at -20oC in small aliquots that
should be discarded after the second cycle of freezing or thawing. During long term storage at
-20oC, small amounts of water evaporate and then freeze on the walls of the vial. To
minimize changes in concentration, vials containing dNTP solutions should be centrifuged,
after thawing, for a few seconds in a micro centrifuge.
4. Divalent cations
All thermostable DNA polymerases require free divalent cations- usually Mg2+ for
activity. Some polymerases will also work, albeit less efficiently with buffers containing
Mn2+. Calcium ions are quite ineffective. Because dNTPs and oligonucleotides bind Mg2+,
the molar concentration of the cation must exceed the molar concentration of phosphate
groups contributed by dNTPs and primers. It is therefore impossible to recommend a
concentration of Mg2+ that is optional in all circumstances. Although a concentration of 1.5
mM of Mg2+ is routinely used, increasing the concentration of Mg2+ to 4.5 mM or 6mM has
been reported to decrease nonspecific priming in some cases and to increase it in others. The
optimal concentration of Mg2+ must therefore must be determined empirically for each
combination of primers and template. The preparations of template DNA should not contain
significant amount of chelating agents (like EDTA or negatively charged ions like PO43-),
which can sequester Mg2+.
5. Monovalent cations
Standard PCR buffer contains 50mM KCl and works well for amplification of
segments of DNA >500bp in length. Raising the KCl concentration to ~70-100mM often
improves the yield of shorter DNA segments.
6. Template DNA:
Template DNA containing target sequences can be added to PCR in single or double
stranded form. Closed circular DNA templates are amplified slightly less efficiently than
linear DNAs. All though the size of the template DNA is not critical, amplification of
sequences embedded in high molecular weight DNA(>10kb) can be improved by digesting
the template with a restriction enzyme that doesn't cleave within the target sequence.
When working at its best, PCR requires only a single copy of target sequence as template.
More typically, however, several thousand copies of the target DNA are seeded into the
reaction. In the case of mammalian genomic DNA,upto 1g of DNA is utilized per reaction,
an amount that contains ~3*105 copies of a single-copy autosomal gene. The typical amounts
of yeast, bacterial and plasmid DNAs used per reaction are 10g, 1g and 1pg respectively.
The cycling reactions :
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is
done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a
very short time.
1. Denaturation at 94C :
During the denaturation, the double strand melts open to single stranded DNA, all enzymatic
reactions stop (for example : the extension from a previous cycle).
2. Annealing at 54C :
The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly
formed and broken between the single stranded primer and the single stranded template. The
more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of
double stranded DNA (template and primer), the polymerase can attach and starts copying the
template. Once there are a few bases built in, the ionic bond is so strong between the template
and the primer, that it does not break anymore.
3. extension at 72C :
This is the ideal working temperature for the polymerase. The primers, where there are a few
bases built in, already have a stronger ionic attraction to the template than the forces breaking
these attractions. Primers that are on positions with no exact match, get loose again (because
of the higher temperature) and don't give an extension of the fragment. The bases
(complementary to the template) are coupled to the primer on the 3' side (the polymerase
adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added
complementary to the template)
Fig 6 : PCR chain reaction
Agarose gel electrophoresis is the easiest and most popular way of separating and
analyzing DNA. Here DNA molecules are separated on the basis of charge by applying an
electric field to the electrophoretic apparatus. Shorter molecules migrate more easily and
move faster than longer molecules through the pores of the gel and this process is called
sieving. The gel might be used to look at the DNA in order to quantify it or to isolate a
particular band. The DNA can be visualized in the gel by the addition of ethidium bromide.
Agarose is a polysaccharide obtained from the red algae Porphyra umbilicalis. Its
systematic name is (14)-3,6-anhydro-a-L-galactopyranosyl-(1 3)--D
galactopyranan. Agarose makes an inert matrix. Most agarose gels are made between 0.7%
and 2% of agarose. A 0.7% gel will show good separation for large DNA fragments (5-10kb)
and a 2% gel will show good resolution for small fragments with size range of 0.2-1kb. Low
percentage gels are very weak (Note:- it may break when you lift them) but high percentage
gels are usually brittle and do not set evenly. The volume of agarose required for a minigel
preparation is around 30-50ml and for a larger gel, it is around 250ml.
Fig7: Structure of agarose
Buffers
Several different buffers have been recommended for electrophoresis of DNA. The
most commonly used buffers are Tris-acetate-EDTA (TAE) and Tris-borate-EDTA( TBE).
The migration rate of DNA fragments in both of these buffers is somewhat different due to
the differences in ionic strength. These buffers provide the ions for supporting conductivity.
Fig 8:The gel with UV illumination: DNA stained with ethidium bromide appears as glowing
orange bands.
DNA as well as RNA are normally visualized by staining with ethidium bromide,
which intercalates into the major grooves of the DNA and fluoresces under UV light. The
ethidium bromide may be added to the agarose solution before it gels, or the DNA gel may be
stained later after electrophoresis. Destaining of the gel is not necessary but may produce
better images. Other methods of staining are available; examples are SYBR
Green, GelRed, methylene blue, brilliant cresyl blue, Nile blue sulphate, and crystal violet.
SYBR Green, GelRed and other similar commercial products are sold as safer
alternatives to ethidium bromide as it has been shown to be mutagenic in Ames test, although
the carcinogenicity of ethidium bromide has not actually been established. SYBR Green
requires the use of a blue-light transilluminator. DNA stained with crystal violet can be
viewed under natural light without the use of a UV transilluminator which is an advantage,
however it may not produce a strong band.
When stained with ethidium bromide, the gel is viewed with an ultraviolet (UV)
transilluminator. Standard transilluminators use wavelengths of 302/312-nm (UV-B),
however exposure of DNA to UV radiation for as little as 45 seconds can produce damage to
DNA and affect subsequent procedures, for example reducing the efficiency
of transformation, in vitro transcription, and PCR. Exposure of the DNA to UV radiation
therefore should be limited. Using a higher wavelength of 365 nm (UV-A range) causes less
damage to the DNA but also produces much weaker fluorescence with ethidium bromide.
Where multiple wavelengths can be selected in the transillumintor, the shorter wavelength
would be used to capture images, while the longer wavelength should be used when it is
necessary to work on the gel for any extended period of time.
The transilluminator apparatus may also contain image capture devices, such as a digital or
polaroid camera, that allow an image of the gel to be taken or printed.
COMPETENT CELL
Competent cells are E. coli cells that have been specially treated to transform
efficiently. There are two types of competent cells: chemically competent and
electrocompetent. If plasmid is simply added to E. coli, nothing happens! The cells must be
competent.
The transformation storage solution (TSS) buffer method, competence is induced by
polyethylene glycol (PEG). This technique is relatively simple and does not require heat
shock. Competence of bacterial cells is induced by the addition of low concentrations of
divalent cations Mg2+ and dimethyl sulfoxide (DMSO). PEG helps in shielding the negative
charges on the DNA molecule and host cell membrane; thus, repulsion between them is
reduced. The pH of the buffer is maintained at slightly acidic conditions to increase the cells
viability as well as transformation efficiency up to 107108 .
PEG is a polyether compound having many functions. In this case, it helps in
shielding the negative charges present on the DNA and host cell membrane. This results in
lowering of repulsion. In addition, PEG, being a larger molecule, coordinates with the water
molecules present in the bacterial suspension. This results in the increased concentration and
bioavailability of the plasmid DNA to pass through the membrane of a bacterial competent
cell. In other words, a highly effective plasmid concentration results in a more effective DNA
transformation into bacteria.
TRANSFORMATION
Bacterial transformation may be referred to as a stable genetic change brought about
by the uptake of naked DNA to increase DNA quantity; competence refers to the state of
being able to take up exogenous DNA from the environment.
Artificial competence can be induced in laboratory procedures that involve making
the cell passively permeable to DNA by exposing it to conditions that do not normally occur
in nature.
The surface of bacteria such asE. Coli is negatively Charged due to
phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively
charged. One function of the divalent cation therefore would be to shield the charges by
coordinating the phosphate groups and other negative charges, thereby allowing a DNA
molecule to adhere to the cell surface.
DNA entry into E. coli cells is through channels known as zones of adhesion or
Bayers junction, with a typical cell carrying as many as 400 such zones. Their role was
established when cobalamine was found to competitively inhibit DNA uptake. Another type
of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is
envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by
Ca ions. It is suggested that exposing the cells to divalent cations in cold condition may also
change or weaken the cell surface structure, making it more permeable to DNA. The heat-
pulse is thought to create a thermal imbalance across the cell membrane, which forces the
DNA to enter the cells through either cell pores or the damaged cell wall.
The lac operator, a sequence downstream of the site where RNA polymerase binds, is
normally occupied by a tetrameric protein called the lac repressor, that is the product of the
lacI gene. This is the state of affairs in the absence of lactose. As long as the repressor is
bound to the operator site, RNA polymerase cannot bind the promoter, and lac Z cannot be
transcribed. If lactose is now added, it binds to the repressor, causing conformational changes
in the latter, so that it no longer binds the operator site. This frees up the promoter for RNA
polymerase binding, leading to transcription of lac Z, and production of beta galactosidase.
In PQE 30, we have inserted the lac operators equence next to the T5 promoter, to
control the expression of the ESAT 6 gene. Instead of lactose, used a chemical analog,
isopropylthiogalactoside(IPTG), that is not metabolized by E. coli, as an inducer. This
inducer binds to the lac repressor, just as lactose would, and turns on the transcription of
ESAT 6. Normal E. Coli cells make some lac repressor, which is encoded by the bacterial lac
I gene. In our experiments we use a strain of E. coli in which the lac I gene has a mutant
promoter that leads to the production of about ten times as much lac repressor as usual.This
mutant lac gene is called lacI q. Strains bearing the lacI q mutation are ideal for expressing
genes under lac operator control, because they make a lot of repressor,thus ensuring that the
operator site is always occupied by the repressor until the inducer is added. For us, this means
that the ESAT 6 gene will remain turned off until we choose to turn it on by adding IPTG.
SDS PAGE
Silver Staining
Usually Silver Staining has better sensitivity than Coomassie Brilliant Blue Staining
and hence is preferred. It raises the sensitivity from microgram level to nanogram level of
protein.
In silver staining, proteins are first fixed onto the gel in acidic conditions where
protonation occurs. Excess acid is then removed by methanol.The gel is then sensitized using
sodium thiosulphate which exposes various functional groups like COOH, -NH3 etc on the
proteins. Then silver nitrate is added wherein Ag ions go n bind to the protein. After
providing alkaline conditions, Ag ions get reduced to metallic silver and black or brown
colored protein band appear on the gel.The reaction is then stopped by providing acidic
conditions.
The 6xHis affinity tag facilitates binding to Ni-NTA. Using pQE vectors it can be
placed at the C- or N-terminus of the protein of interest. It is poorly immunogenic, and at pH
8.0 the tag is small, uncharged, and therefore does not generally affect secretion,
compartmentalization, or folding of the fusion protein within the cell. In most cases, the
6xHis tag does not interfere with the structure or function of the purified protein as
demonstrated for a wide variety of proteins, including enzymes, transcription factors, and
vaccines.
A further advantage of the 6xHis tag is that it allows the immobilization of the protein
on metalchelating surfaces such as Ni-NTA HisSorb Strips or Plates and therefore simplifies
many types of protein interaction studies. In addition, AntiHis Antibodies can be used for
detection. Ni-NTA technology Immobilized-metal affinity chromatography (IMAC) was first
used to purify proteins in 1975 (Porath et al. 1975) using the chelating ligand iminodiacetic
acid (IDA, Figure 4). IDA was charged with metal ions such as Zn2+, Cu2+, or Ni2+, and
then used to purify a variety of different proteins and peptides (Sulkowski 1985). IDA has
only 3 metal-chelating sites and cannot tightly bind metal ions. Weak binding leads to ion
leaching upon loading with strongly chelating proteins and peptides or during wash steps.
This results in low yields, impure products, and metal-ion contamination of isolated proteins.
Fig: 9
Fig :11
Binding of tagged proteins to Ni-NTA resin is not conformation-dependent and is not
affected by most detergents and denaturants (Table 4, page 74). The stability of the 6xHis
Ni-NTA interaction in the presence of low levels of ME (up to 20 mM) in the lysis buffer can
be used to prevent the copurification of host proteins that may have formed disulfide bonds
with the protein of interest during cell lysis. Detergents such as Triton X-100 and Tween 20
(up to 2%), or high salt concentrations (up to 2 M NaCl) ,also have no effect on binding, and
may reduce nonspecific binding to the matrix due to nonspecific hydrophobic or ionic
interactions. Nucleic acids that might associate with certain DNA and RNA-binding proteins
are also removed without affecting the recovery of the 6xHis-tagged protein.
Western blotting
Gel electrophoresis
Western blot uses two different types of agarose gel: stacking and separating gel. The
higher, stacking gel is slightly acidic (pH 6.8) and has a lower acrylamide concentration
making a porous gel, which separates protein poorly but allows them to form thin, sharply
defined bands. The lower gel, called the separating, or resolving gel, is basic (pH 8.8), and
has a higher polyacrylamide content, making the gel's pores narrower. Protein is thus
separated by their size more so in this gel, as the smaller proteins to travel more easily, and
hence rapidly, than larger proteins.
The proteins when loaded on the gel have a negative charge, as they have been denatured by
heating, and will travel toward the positive electrode when a voltage is applied. Gels are
usually made by pouring them between two glass or plastic plates, using the solution
described in the protocol section. The samples and a marker are loaded into the wells, and the
empty wells are loaded with sample buffer. The gel is then connected to the power supply
and allowed to run. The voltage is very important, as a high voltage can overheat and distort
the bands.
Blotting
(1) the close contact of gel and membrane to ensure a clear image and
(2) the placement of the membrane between the gel and the positive electrode.
The membrane must be placed as such, so that the negatively charged proteins can
migrate from the gel to the membrane. This type of transfer is called electrophoretic transfer,
and can be done in semi-dry or wet conditions. Wet conditions are usually more reliable as it
is less likely to dry out the gel, and is preferred for larger proteins.
The membrane, the solid support, is an essential part of this process. There are two
types of membrane: nitrocellulose and PVDF. Nitrocellulose is used for its high affinity for
protein and its retention abilities. However, it is brittle, and does not allow the membrane to
be used for reprobing. In this regard, PVDF membranes provide better mechanical support
and allow the blot to be reprobed and stored. However, the background is higher in the PVDF
membranes and therefore, washing carefully is very important.
The membrane is then detected using the label antibody, usually with an enzyme such
as horseradish peroxidase (HRP), which is detected by the signal it produces corresponding to
the position of the target protein. This signal is captured on a film which is usually developed
in a dark room.
Quantification
It is very important to be aware that the data produced with a western blot is typically
considered to be semi-quantitative. This is because it provides a relative comparison of
protein levels, but not an absolute measure of quantity. There are two reasons for this; first,
there are variations in loading and transfer rates between the samples in separate lanes which
are different on separate blots. These differences will need to be standardized before a more
precise comparison can be made. Second, the signal generated by detection is not linear
across the concentration range of samples. Thus, since the signal produced is not linear, it
should not be used to model the concentration.
MATERIALS AND MEATHODS
3.1 Confirmation of presence of ESAT-6 gene insert of M.tuberculosis in
the plasmids by polymerase chain reaction (PCR) :
PCR was carried out to confirm the presence of ESAT-6 insert in the plasmids stored at - 20.
Materials
1. Forward primer of ESAT-6
Sequence of forward primer: -
5'TAACGGAGCAAAGGATCCACAGAGCAGCAG3'
2. Reverse primer of ESAT-6
Sequence of reverse primer:
5'TTCTACGCGAGGATCCAAGCTTCTATGCGAACATCCC3'
3. Master mix :
Taq DNA polymerase ,dNTP's( containing 1 mM of each dATP, dTTP, dCTP,
dGTP) ,MgCl2 ,Buffer
4. Template : Recombinant PQE 30 plasmid with ESAT-6 insert.
5. Positive control DNA:M. tuberculosis H37Rv genomic DNA (500 ng)
6. PCR machine: Cycler Gradient Eppendroff
Set up For 1 reaction
Constituents Amount
Master mix 3 l
Forward primer 1 l
Reverse primer 1 l
Distilled water 19 l
Template 1 l
Total volume 25 l
Methods:
Twenty five micro-liter of reaction mixture prepared as above and was added to 0.2
ml PCR tubes. PCR was carried out in an Eppendorff thermal cycler to amplify the required
fragment from given PQE 30 plasmid with ESAT-6 insert. PCR consisted of 35 cycles,
each cycle comprising of following steps.
PCR program steps were as follows-
Method:
1. Make 1% agarose- 0.8 gm of agarose dissolve in 40 ml of 1X TAE buffer and boil for 30
sec till agar get completely dissolved.
2. Clean the electrophoresis unit and pore agarose into the gel casting tray, having a gel
comb.
3. After the gel is solidified, add 300 ml of 1X TAE Buffer into it and remove the comb
without disturbing gel.
4. Sample preparation- 2 l of gel loading dye + 5 l of amplified DNA sample, was
carefully loaded into the wells.
5. Run the gel at 70 V till band reaches the end of the gel and was observed under UV
transilluminator
Materials:
1. E.coli M15[pREP4] cells preserve as a frozen glycerol stock in liquid nitrogen.
2. LB medium
LB agar 1.75 gm of LB agar in 50 ml of D/W
LB broth- 2 gm of LB broth in 100 ml ofD/W
3. TSS buffer ( 50 ml)
1 PEG 8000 5 gm
2 1M MgCl2 1.5 ml
3 DMSO 2.5 ml
4 LB broth By using LB make total
volume upto 50 ml
Filter sterilized with 0.22 m filter.
5. Crushed ice
6. 370 C shaker for incubation
7. Centrifuge
Method:-
DAY 1
1. Take a loopful of frozen E. coli M15[pREP4] cells from vial , and streak it out on LB
agar containing 25 g/ml kanamycin.
2. Incubate at 37C overnight.
DAY 2
3. Pick up a single colony and inoculate 10 ml of LB containing kanamycin (25 g/ml).
Grow overnight at 37C in shaker waterbath .
DAY 3
4. Add 0.5 ml overnight culture to 50 ml prewarmed LB broth containing 25 g/ml
kanamycin in a 250 ml flask, and shake at 37C until anOD of 0.6 at 600nm is
reached (approximately 90120 min).
5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom
centrifuge tube.
6. Collect the cells by centrifugation at low speed (5 min, 4000 x g, 4C).
7. Discard the supernatant carefully. Always keep the cells on ice.
8. Resuspend the cells gently in cold (4C) TSS buffer (The volume off TSS to use is
10% of the culture volume that is spun down)
9. Prepare aliquots of 100200 l in sterile microcentrifuge tubes and freeze in liquid
nitrogen or a dry-iceethanol mix. Store the competent cells at 70C.
Method: -
1. Thaw an aliquot of frozen competent E. coli M15[pREP4] cells on ice.
2. Gently resuspend the cells and transfer 100 l of the cell suspension into the
microcentrifuge tube with the 1 l of recombinant plasmid, mix carefully, and keep it on
ice for 60 min.
3. Add 0.9 ml LB broth to the cells and incubate for 60min at 37C in shaker waterbath
(Shaking increases transformation efficiency).
4. Plate out 50, 100, and 200 l aliquots on LB-agar plates containing 25 g/ml kanamycin
and 100 g/ml ampicillin. Incubate the plates at 37C overnight.
Method:
1. Take 0.1 ml of the overnight culture into a microfuge tube. Centrifuge at 12000 rpm
for 1 min at 4C .
2. Remove medium by aspirations, leaving the pellets as dry as possible.
3. Resuspend the pellet in200 l of ice cold buffer PA by gental vortexing.
4. Add 1 l RNAse A stock to the cell suspension.
5. Add 200 l of buffer PB.
6. Close the tube tightly and mix the contens by inverting the tube slowly 4 times.keep
the tube on ice.
7. Add 300 l of ice cold buffer PC. Close the tube tightly and mix the contents by
inverting the tube slowly for 4 times.
8. Centrifuge at 13000 rpm for 5 min at 4C to get a white pellet aspirate the entire
supernated carefully and load onto spin column in 2 ml collection tube. If pellet get
disturb reapet the step again.
9. Close the cap and centrifuge at 9000 rpm for 1 min.
10. Discard the filtrates and place the column in same collection tube.
11. Add 500 l of PD buffer and centrifuge at 9000 rpm for 1 min. Discard the filtrates
12. Add 500 l of PE buffer and centrifuge at 13000 rpm for 3 min
13. Place the column in claen 1.5 ml microfuge tube and discard the collection tube.
14. Add 50 l of buffer PF prewarm at 65 C, incubate for 2-3 min at room temperature
and centrifuge at 10000 rpm for 3 min.
15. The eluted solution can be used directly for PCR or restriction digestion or can stored
at - 20C for further use.
3.6 Induction for overexpression of ESAT-6 protein using IPTG
Materials:
4. IPTG ( 1M)
238 mg /ml in H2O , sterile filter, store in aliquots at -20C.
Method: -
1. Pick a single colonies of tranformants into 5 ml of LB medium containg ampiciline
(100 ug/ml) and kanamycine ( 25 g/ml).
2. Grow the culture overnight at 37C. Shaking
3. Transfer 0.5 ml overnight culture into 50 ml of LB broth containg ampiciline (100
g/ml) and kanamycine( 25 g/ml).and grow at 37C for approx 2-3 hrs with vigorous
shaking ,until the O.D. 600 is 0.5-0.6 .
4. Before induction remove 1 ml of culture and Mark as control 0 hr.
5. In remaining culture induce expression by adding IPTG to final concentration of 1 mM.
6. Incubate at 37C shaker.
7. Remove 1 ml of induced culture after each 1 hr time interval ( label as 1 hr , 2hr, 3hr, 4
hr)
Method:
1. Cell pellet was weighed and was resuspended in lysis buffer ( for 1gm of pellet ,add 4
ml of lysis buffer )
2. Tube was incubated on ice for 20 mins
3. Suspension was homogenized and sonicated briefly to decrese the viscosity.
4. Lyset was centrifuge ( 4000 rpm at 4C for 5 mins)
5. Supernated was save for SDS PAGE analysis, pellet used for harvested cells under
denaturing condition
Materials:
Pellet fraction obtained in above step after lysis.
Lysis buffer containing 8M urea
Method :
1.Lysed cell pellet was weighed and was resuspended in urea containing lysis buffer.
2.Tube was incubated at 4C for 20 mins
3.Lyset was homogenized and centrifuged 4000 rpm at 4C for 5 mins.
4.Supenated was saved for SDS PAGE analysis.6
4. 10% SDS
Add 1gm of SDS in 10 ml of D/W.
5. 30 % acralamide gel
3 gm of acralamide in 30 ml of D/W
Method:
Preparation of gel mix
4% stacking gel mix
Composition Amount
Distilled water 3.675 ml
30% acralamide gel 0.65 ml
1 M TrisHCl (pH 6.8) 0.625 ml
10% SDS solution 50 l
10% APS solution 38l
TEMED 7.5l
Total volume 5.0 ml
Composition Amount
Distilled water 3.98 ml
30% acralamide gel 3.33 ml
1.5 M Tris HCl (pH 8.8) 2.5ml
10% SDS solution 75 l
10% APS solution 100l
TEMED 7.5l
Total volume 10ml
Sample preparation :
Sample obtained after IPTG induction add lysis buffer contains 8 M urea into it. Sonicate the
sample before use.16l of sample + 4 l of gel loading dye heat it at 70C for 5 min and load on the
gel.
1. Gel casting assembly was set up, leak test was performed and resolving gel mix was
th
poured upto 3/4 of the glass plate.(TEMED and APS being added just before
pouring the gel)
2. A thin layer of water was overlaid.
3. On polymerization of the resolving gel, water was wiped off using a blotting paper.
4. Stacking gel mix was then poured and comb was inserted in place.
5. On polymerization the comb was removed and the gel was transferred to the running
assembly.
6. Tank Buffer was added. Samples and the protein marker were prepared by mixing
with loading dye in ratios as required, boiled for around 3 mins and then loaded into
individual wells.
7. The gel was run at 50 V.
Materials:
1. Fixative (100 ml)
methanol - 45 ml
Glycial acetic acid -10ml
H2O- 45 ml
Formaldehyde- 75 l
3. Sensitizing solution(100ml)
Na2S2O3 - 0.02gm + 100 ml D/W
4. Staining solution(100ml)
AgNO3 -0.02 GM + 76l of formaldehyde + 100 ml of D/W
6. terminating solution.(100ml)
Glyacial acetic acid 12ml + D/W 88 ml
Method:
1. Wash the gel with distilled water twice ( 10 mins each on shaker)
2. Keep the gel in 20% Methanol(washing solution) ( on shaker at R.T. for 20 min)
3. Add sensitizing solution for 2 min.
4. Wash the gel twice with D/W for 2 Min
5. Add chilled staining solution and incubate in dark for 20 mins.
6. Wash with D/W twice (1 min each)
7. Add developing solution on it till bands are developed
8. After developing the band add terminating solution (12%) on it to terminate the
reaction.
Material
Lysis buffer (1 liter):
50 mM NaH2PO4 = 6.90 g NaH2PO4H2O (MW 137.99 g/mol)
300 mMNaCl = 17.54 g NaCl (MW 58.44 g/mol)
10 mM imidazole = 0.68 g imidazole (MW 68.08 g/mol)
Adjust pH to 8.0 using NaOH.
Method:
1. Wash Ni-NTA column with lysis buffer add 2 ml Ni-NTA slurry with lysis buffer.
2. Add cells which is suspended in lysis buffer after IPTG induction
3. Mix it with Ni-NTA slurry and keep on rotary shaker at RT for 2 hrs
4. After incubation add slurry into column containing filter disc and collect flow
through.
5. After selling the slurry into the column. Add 2ml if washing solution onto it and
collect the wash through.
6. Elute the solution with elution buffer , Collect the filtrates and subjected to SDS
7. PAGE for analysis of purified protein.
8. And gel further subjected to silver staining and Western blotting.
Materials:
2. Transfer buffer(10X)
Tris 30gm + glycine 144gm
Dissolved in D/W and make volume up to 100Oml and store at 4C
To make 1X from 10 X=
50 ml of 1X transfer buffer + 100 ml Methanol + 300 ml of D/W
Miscellaneous
1. Nitrocellulose membrane
2. 3 MM Whatmann paper
3. Western blotting electrophoretic apparatus
4. Transfer chember
5. Power pack
6. Shaker
7. X- ray developing system
Method :
1. The nitrocellulose membrane (0.2m) and 3MM Whatmann paper were cut according
to the gel size and soaked in 1X transfer buffer
2. The assembly was arranged: sponge- Whatmann paper- gel nitrocellulose membrane
Whatmann paper sponge.
3. It was place in transfer chamber filled with transfer buffer.
4. Transfer was carried out at 90V for 1 hour.
5. The assembly was removed. The nitrocellulose membrane was wash with 1X TBS-T
6. Membrane was stained with ponceau S solution ( 0.1% ponceay S(w/v) in 5% acetic
acid (w/v).to check whether the transfer occur or not.
7. The membrane was wash with the 1X TBS-T till the stain is removed.
8. It was put in blocking solution( 5% skimmed milk ) n shaker for 15-30 mins.
9. Then it was wash with 1X TBS-T for 2 mins
10. 1:2000diluted primary antibody in 1X TBS-T with 2.5% skimmed milk was added
and it was incubated for 5 hrs.
11. The membrane wash with 1XTBS-T on shaker at R.T 3 times ( 15 min each)
12. Add 1:4000 diluted secondayantibody in 1X TBS-T with 2.5% skimmed milk was
added and it was incubated for 2 hrs.
13. The membrane was wash with the 1X TBS-T onshaker at R.T 3 times( 15 mis each)
14. Membrane was developed using chemi-luminescence kit.
RESULTS
Competent cells of E. coli M15 were prepared using TSS method and transformation of
recombinant PQE 30 was carried out. To check the viability of the compitant cells,
aliquots were spread onto LB agar plate with Kanamycine. Fig 2__ shows that the
competence celss are live and are not dead due to treatment.
Fig 2 : Competence cells formed using TSS method
The competent cells were further subjected to transformation with plasmids which showed
positive results and thus presence of ESAT-6 gene. To confirm the transformation,
aliquots were spread on LB plate containing kanamycine and ampicilline.
Fig 3 . E. Coli containing the recombinant plasmid (ESAT-6 gene insert in PQE 30
plasmid)
Fig 5 : SDS PAGE gel showing the unininduced and IPTG induced E.coli M15 lysate
Lane 1:IPTC induced pellet ( 3 hours )
Lane 2: IPTG induced supernatant ( 3 hours )
Lane 3 :IPTC induced pellet( 2 hours )
Lane 4 :IPTG induced supernatant( 2 hours )
Lane 5 : protein ladder
Lane 6 : IPTG induced supernatant( 4 hours )
Lane 7 :IPTC induced pellet( 4 hours )
Lane 8 : non induced lysate
Lane 9 :IPTC induced pellet( 1hour)
Lane 10: IPTG induced supernatant( 1hour )
After confirming presence of ESAT-6 protein in lysate by SDS- PAGE and silver
staining method,recombinant protein was bulk purified using Ni-NTA column
chromatography. Oveexpresssed protein was eluted using appropriate elution buffer
containing imidizole SDS-PAGE of eluted samples on 15% gel was carried out in order to
analyze the purity of the eluted fractions . Fig 6 and 7 shows presence of single bands
indicating presence of ESAT-6 antigen.
Tuberculosis (TB) remains a major global health problem. Human Tb is the most frequent
cause of death from a single infectious agent , being responcible for 8 million new cases and
approximately 1.6 million death annually. Given the infectious nature of pulmonary
tuberculosis fast and accurate diagnosis is an important element of TB treatment and control.
Current serological test for diagnosis of tuberculosis Are simple and inexpensive but have
certain limitations with respect to sensitivity and specificity. Sensitivity of these sero-
diagnostic test can be improved only with the use of purified antigens. These purified
antigens can also prove as potential candidates for useas subunit vaccines. Thus,
identification of such purified antigens of mycobacterial tuberculosis which are involved in
recognition by host immune system is required to improve the efficiency of serodiagnostic
test.
The main aim of the study was to overexpress and purification of recombinant protein and its
confirmation of Western Blot. This antigen is abundant and constitutes a major component in
culture filtrate as wall as in cell wall preparation. CFP 10 and ESAT 6 are interesting
candidates for the diagnostics of tuberculosis. These protein elicit a strong T-Cell response in
animal models of TB and human active TB infection. The ability of a protein to detect
antibody present during subclinical disease is an important as the sensitivity of the protein in
detecting antibody formed during active tuberculosis , since therapy for it can prevent the
development of active disease . the gene coding ESAT 6 is located in the RD1 region of the
M.TB which is responsible for virulence. During the course of passaging to obtained
attenuated strain, this region is lost as a result of which required immunity against TB is not
conferred by the conventional BCG vaccine.
Our object in this study was to overexpress early secretary antigenic target protein of M
tuberculosis in E.coli M15. The DNA segment corresponding to gene encoding ESAT 6
protein of M. tuberculosis was successfully amplified by PCR, cloned and reconbinat clones
were screened by PCR for confirmation of the insert. the cloned ESAT 6 were induced with
IPTG for overexpression of recombinant protein and SDS pahe followed by silver staing of
the gel was performed these expressed protein were purified by affinity chromatography in
Ni-NTA column run the gel and performed western blottingand then reacted with anti-
penta- his antibody followd by rabbit antimous antibody HRP conjugate for confiomation.
After purification and its cobfirmation the prurified antigen can be used further for the
detection of antibody against it in sera of Tb patient and also can be used after immunological
characteristics in the basic research for understanding the host parasite infection.
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