A novel protein from helicobacter pylori with a potential role in gastroduodenal diseases

pylori and gastric mucus layer The human stomach is protected by the gastric acid of a pH around 2.0 while a viscous layer of mucus acts as the protective barrier for the underlying ga

1.1 Helicobacter pylori and gastroduodenal diseases

Helicobacter pylori is a gram-negative, spiral-shaped microaerophilic bacterium that

colonizes the human gastric mucosa Since the successful isolation of H pylori by

Marshall and Warren in 1983, doors have been opened for scientists to study the

association of H pylori with various gastroduodenal diseases Persistent colonization of

H pylori in human gastrointestinal tracts has been closely linked to gastric diseases

ranging from gastritis, non-ulcer dyspepsia and peptic ulcer to the increased risks of

gastric cancer (Buck et al., 1986; Dunn et al., 1997) Being a major human gastric

pathogen, H pylori infects more than half of the world’s population and has been causing

gastric diseases worldwide (Blaser et al., 1994; Uemura et al., 2001)

For the past two decades, great effort has been focused on the study of H pylori

with respect to its bacteriology, physiology, genetics, pathogenesis and epidemiology of

infection (Rothenbacher & Brenner, 2003) Treatment of peptic ulcer disease has been

developed (Graham et al., 1992) However, current therapies remain to be improved as

the organism is perfectly adapted to the ecological niche in the gastric mucosa and

currently available antibiotics are not specifically designed to be active in the stomach

(Sherwood et al., 2002; Schreiber et al., 2004)

1.2 Characteristics of H pylori

Two morphological forms were observed in H pylori: spiral and coccoid (Hua &

Ho, 1996) The spiral-shaped H pylori is the active form capable of colonization and

infection (Dunn et al., 1997) On the other hand, the coccoid form is considered as viable

but non-culturable (Van et al., 1994) and has been considered as the resting state of the

bacterium (Benaissa et al., 1996) Under unfavorable conditions such as depletion of

nutrients, addition of antibiotics or stress stimuli (low pH or high temperature),

morphological conversion from spiral to coccoid can be observed in in vivo culture

(Catrenich & Makin, 1991) However, the resuscitation from coccoid to spiral has not

been reported in in vitro conditions Therefore, controversies remain among researchers

as some regarded coccoids as dead bacterial cells (Kusters et al., 1997; Enroth et al.,

1999) while others believed that coccoids are viable but non-culturable (Hua & Ho, 1996;

Zheng et al., 1999; Saito et al., 2003)

The spiral form of H pylori expresses a great number of proteins that participate in

various bacterial metabolic activities such as cell survival and proliferation, adhesion,

colonization and transportation of macromolecules In contrast, the coccoid expresses

significantly less proteins related to the basic metabolism such as cell respiration,

maintaining cellular integrity and DNA synthesis (Kusters et al., 1997; Narikawa et al.,

1997; Costa et al., 1999) It is widely agreed that the spiral form is mainly responsible for

the pathogenesis of H pylori infection (Dubois, 1995) In contrast, it has been suggested

that the dormant coccoid form may be involved in the transmission of H pylori infection

(Hua & Ho, 1996; Zheng et al., 1999; Andersen et al., 2000; Ng et al., 2003)

1.3 Pathogenesis of H pylori

Several proteins have been identified in H pylori to be associated with its virulence

and pathogenesis The most intensively studied virulence factors are cytotoxin-associated

immuno-dorminant protein (CagA), vacuolating toxin A (VacA), adhesins, flagella,

urease and heat shock proteins (HSPs) These factors act independently from each other

in the course of H pylori infection but are indispensable for bacterial pathogenesis (Prinz

et al., 2003)

Among the various virulence factors, adhesins are important for mediating

receptor-ligand recognition in the initial interaction between H pylori and host Many H pylori

proteins have been identified as adhesins specific for interaction with particular ligands of

the host (Aspholm et al., 2004) These include blood-group-antigen-binding adhesion

(BabA) which is an adhesin of H pylori interacting with the blood group antigen-Lewis

antigen on gastric epithelial cells (Ilver et al., 1998; Hennig et al., 2004); Sialic

acid-binding adhesion (SabA) that is responsible for the acid-binding of H pylori to sialyl-Lewis x

antigens in gastric epithelium in humans (Mahadavi et al., 2002); OipA (outer

inflammatory protein) and HopZ (homologue of porin) which are associated with the

adhesion and colonization of H pylori in vitro and in vivo, respectively (Yamaoka et al.,

2002)

The sheathed flagellum is responsible for the motility of H pylori which is

necessary for bacterial survival in the viscous mucus layer (Josenhans et al., 1995) while

surface localized urease is an enzyme served to maintain a neutral pH microenvironment

for the survival of H pylori in the acidic stomach (Eaton et al., 1991; Perez-Perez et al.,

1992; Clyne et al., 1995) CagA and VacA have been proven to be two major virulence

factors of H pylori, of which CagA protein can be translocated into the epithelial cells to

trigger a cascade of signal transduction pathways (Segal et al., 1999; Hirata et al., 2004)

while VacA is known for its ability to induce cytoplasmic vacuole formation in various

eukaryotic cells (Telford et al., 1994; Cover et al., 2005) HSP is another group of

virulence factor essential for maintaining the normal functions of other H pylori proteins

and assisting its survival in the stomach (Kamiya et al., 1998)

1.4 Interaction between H pylori and gastric mucus layer

The human stomach is protected by the gastric acid of a pH around 2.0 while a

viscous layer of mucus acts as the protective barrier for the underlying gastric epithelium

(Vinall et al., 2002) The main component of the mucus is a high molecular weight

glycoprotein known as mucin, which has a long linear structure consisting of subunits

joined by disulfide bonds (Thomsson et al., 2002) The subunit of mucin contains a

peptide core that is interspersed among clusters of oligosaccharide side chains (Stanley et

al., 1983; Karlsson et al., 1996) The diversity of mucin oligosaccharides has been found

to provide multiple receptors for bacterial lectins of invading pathogens (Karlsson et al.,

1989 & 1995) Furthermore, experimental evidence has illustrated that the adherence to

mucin occurs in a number of pathogens such as Pseudomonas aeruginosa, Candida

albicans and Staphylococcus aureus (Hoffman et al., 1993; Bruce et al., 1995; Shuter et

al., 1996) In the case of H pylori, it is believed that attachment to the mucus layer has

been established between the bacteria and host before its colonization at the epithelium

and specific adhesins could exist in mediating such interaction (Linden et al., 2002)

Kalpana (2003) described a protein from H pylori that has the ability of binding to mucin

in in vitro assays which has been identified to be a hypothetical protein of H pylori

coded as HP0049 in the genome of H pylori ATCC 26695 (Tombs et al., 1997) It is

therefore interesting to characterize the functions of this novel protein and explore its

possible role in the pathogenesis of H pylori related gastroduodenal diseases

1.5 Objectives of study

This study aims to characterize this hypothetical protein HP0049 and examine its

functions in H pylori The main goals of the project are to clone and express the gene

encoding HP0049 followed by characterization of the recombinant protein In the process,

the recombinant protein will be employed to raise specific antibody, which will be used

for sub-cellular localization of the protein in H pylori by transmission electron

microscopy (TEM) Biochemical and physiological studies will be carried out to explore

the probable role of the hypothetical protein HP0049 in H pylori pathogenesis

2.1 Characteristics of H pylori

2.1.1 Isolation and culturing of H pylori

Research in H pylori began in earnest after its isolation in 1983 by Marshall and

Warren Helicobacters belong to a new genus of bacteria, mainly inhabiting along the

interface of mucosa and gastric epithelial cells of mammals H pylori NCTC 11637 is the

type species of the genus, Helicobacter It is gram-negative, microaerophilic,

spiral-shaped, flagellated and urease positive bacterium It is a nutritionally fastidious

microorganism which is able to form about 1 mm transparent colonies on enriched agar

plates supplemented with 5-10% horse blood after 3-5 days of incubation (Goodwin &

Worsley, 1993) H pylori is also an oxygen sensitive bacterium that strictly grows in the

presence of 5-10% carbon dioxide at 35-37˚C under humidified conditions (Goodwin et

al., 1986) The organism can proliferate in both non-selective and selective media

supplemented with antibiotics (Goodwin & Worsley, 1993; Westblom et al., 1991)

whereby the latter are widely used for the isolation of H pylori from biopsy samples

H pylori can be cultured on solid agar plates as well as in liquid media However,

the growth of H pylori in liquid media (generally enriched with yeast extract and serum)

is comparatively slower than that on the agar plate Nevertheless, broth culture has been

used favourably in the study of its metabolic activities and and physiological properties

(Goodwin et al., 1986; Ho & Vijayakumari, 1993) H pylori has been shown to grow in

brain heart infusion (BHI) broth supplemented with 0.4 % yeast extract and 10% horse

serum under microaerophilic conditions in an efficient and continuous culture system (Ho

& Vijayakumari, 1993)

2.1.2 Morphological features of H pylori

Based on electron microscopy studies, two major morphological forms of the

bacteria were observed: spiral and coccoid In an early study by Benaissa et al (1996),

the conversion from spiral to coccoid via U-shaped transition form was clearly observed

under transmission electron microscopy Thereafter, similar observations of

morphological conversion were reported by other researchers in later studies (Kusters et

al., 1997; Costa et al., 1999) The spiral shaped H pylori is approximately 0.5 µm in

width and 2-3 µm in length, equipped with 4-6 unipolar-sheathed flagella that are

continuous with the outer cell membrane (Wang et al., 2006) It is believed that the spiral

shape of the bacteria and corkscrew movement provided by the sheathed flagella have

enabled the organism to penetrate the viscous gastric mucus layer (Ottemann &

Lowenthal, 2002) The ultra thin sections of H pylori under electron microscopy also

exhibited the typical cell wall structure of gram-negative bacterium that consists of both

outer and inner membrane, with condensed cytoplasm containing nucleoid material and

ribosome (Costa et al., 1999)

On the other hand, the round shaped coccoid forms of H pylori are more or less

regarded as degenerative and dead cells (Kuster et al., 1997) There is another group of

researchers who suggested that the coccoid form is viable but non-culturable (Hua & Ho,

1996; Saito et al., 2003) Substantial variation in cell wall structure, surface protein

profiles and DNA contents were detected during the transition from spiral to coccoid

(Benaissa et al., 1996; Costa et al., 1999), which hints that the two differential forms of H

pylori could contribute different roles during infection

2.1.3 Genetics of H pylori

The complete genome of H pylori strain 26695 was reported by Tomb et al in 1997

It contains 1,667,867 base pairs of nucleotides and 1,590 predicted coding regions A

second complete genome of strain j99 was published in 1999 (Alm et al) Comparison of

the two genomes revealed higher than 97% homology between them at the gene size and

gene order with a limited number of discrete regions that are organized differently When

viewed in a genome wide manner, about 6-7% of the annotated genes are strain specific

and are absent from each other with no homologue in the database (Alm et al., 1999)

The prevalence of H pylori infection varies in different geographical regions,

ethnic backgrounds, socioeconomic conditions and age groups (Covacci et al., 1999),

possibly contributed by the diversified bacterial genotypes of the isolates (Yamazaki et

al., 2005) Unusual heterogeneity has been observed among the genotypes of clinical

isolates and bacterial populations within the infected hosts, and the variation in bacterial

population can be observed in individuals infected with more than one H pylori strain

(Blaser, 1990) The evolution of genotypic diversity in H pylori may have been resulted

from the presence of multiple strains within the same host, as plural cohabitation tends to

favor the occurrence of free intraspecies recombination

2.2 Virulence factors of H pylori

2.2.1 Adhesins of H pylori

Many adhesins have been identified or predicted by the annotation of ORFs (open

reading frames) in the H pylori genome (Tomb et al., 1997; Alms et al., 1999) Most of

them are outer membrane proteins that mediate receptor-ligand interaction between

bacteria and host escorting the adhesion of H pylori onto the mucus layer (Doig et al.,

1992) Blood group A antigen-binding adhesin (BabA) is one such adhesin that has been

extensively studied BabA is encoded by allelic babA2 gene and is involved in the

binding of bacteria to the blood group antigen Lewis b surface epitopes of host (Boren et

al., 1993; Ilver et al., 1998) Another newly identified adhesin is SabA (Sialic

acid-binding adhesion) for acid-binding to sialyl-Lewis x antigens in gastric epithelium in humans

(Mahadavi et al., 2002) The adherence of H pylori to sialylated glycoconjugates

expressed during chronic inflammation contributes to the virulence and the extraordinary

chronicity of H pylori infection (Mahadavi et al., 2002)

The other H pylori proteins reported to be involved in the adherence of H pylori

onto the gastric epithelium are OipA (outer inflammatory protein, HP0638) (Yamaoka et

al., 2002) and HopZ (homologue of porin) (Peck et al., 1999) It was noted that different

numbers of CT dinucleotide repeats are characteristically present in the sequences of

these adhesion genes (oipA, hopZ and sabA), which determine the functional status of

these genes In addition, it was also reported that the inclusion of the CT nucleotide

repeats in the open reading frame would turn on the expression of these genes whereas

the deletion and substitution of those repeats would cease the expression of these genes

(Yamaoka et al., 2002) Interestingly, the functional status (on/off) of oipA, hopZ and

sabA were found to affect the adherence and colonization properties of H pylori

(Yamaoka et al., 2002)

2.2.2 Other pathogenic factors of H pylori

A number of H pylori proteins have been established as virulence factors involved

in its infection of the gastric mucosa Among these factors, two major groups were

classified as either related to adhesion and colonization of H pylori or causing damage to

host cells and benefiting the survival of bacteria in vivo

Among the bacterial proteins that have been reported to be crucial for the adhesion

and colonization of H pylori include flagellins that are responsible for bacterial motility

(Josenhans et al., 1995; Ottemann & Lowenthal, 2002) and urease that neutralizes the

acidic pH (Dunn et al., 1990; Tsuda et al., 1994; Karita et al., 1995) There are also

various catalases and oxidases (Harris et al., 2003) that are enzymes leading to different

biochemical degradations Others include outer membrane proteins with or without

known functions (Yamaoka et al., 2002), phospholipase (Dorrell et al., 1999) and

numerous adhesins that mediate the adhesion of H pylori to different host ligands

Mutations in the genes coding for these adhesive proteins in H pylori have been found to

reduce the adherence capability or colonization ability of the bacteria onto the gastric

mucosa in animals (Evans et al., 2000)

The other major group of virulence factors that causes tissue damage include

vacuolating cytotoxinA (VacA) and cag pathogenicity island (cagPAI) which have been

shown to be related to peptic ulcer and associated with the induction of immune response

of the host (Le’Negrate et al., 2001) In addition, recent studies indicate that heat shock

proteins e.g HSP60 are necessary for the bacteria in combating against the hostile

environment (Spohn et al., 2002)

Findings on H pylori virulence factors showed that the bacteria possess a group of

unique features essential for its successful colonization in the host Adherence to the

gastric mucus layer protects the organism against the hostile acidic environment in the

stomach and thereby allows the bacteria to penetrate and establish persistent colonization

on the gastric epithelium During the course of its infection, H pylori induces various

levels of immune responses in the host which further develop into different types of

gastroduodenal diseases

2.3 H pylori infection and gastroduodenal diseases

It is reported that more than 50% of the world’s population is infected with H pylori

(Blaser, 1990) and the prevalence of H pylori infection varies in different regions, races

and age groups Significant difference in the incidence of H pylori infection has been

noticed between developing and developed countries, with a level as high as 70-90% and

as low as 20-40%, respectively (Bardhan, 1997) In multi-ethnic Singapore, the infection

rates of H pylori in Chinese and Indian populations are 2.5 times higher than that in the

Malays (Epidemiol News Bull, 1996), suggesting the possible effect of racial differences

in and host genetic predisposition to H pylori infection

A prospective study from infancy to adulthood demonstrated that H pylori infection

could be detected at an early stage of life at the age of 10 (Malaty et al., 2002) The

finding indicates that the acquisition of H pylori may occur in childhood by vertical

transmission through intrafamilial route (e.g from parents to children, or grand parents to

grand children) This has also been supported by reports of several other studies (Taneike

et al., 2001; Ng et al., 2003; Roma-Giannikou et al., 2003)

H pylori infection is closely associated with the induction of various gastroduodenal

diseases including gastritis, non-ulcer dyspepsia, gastric ulcer, duodenal ulcer as well as

gastric cancer About 90% of chronic gastritis is caused by H pylori (Sobala et al., 1991)

The association of non-ulcer dyspepsia (NUD) with H pylori has been debated among

researchers (Pieramico et al., 1993) Recent reports showed that dyspeptic symptoms

concurred with the infection of certain virulent H pylori strains (Treiber et al., 2004) H

pylori remains the main etiological agent of peptic ulcer including gastric ulcer (GU) and

duodenal ulcer (DU) (Mauch et al., 1993; Moss & Calam, 1993) while the eradication of

H pylori enhances the healing process of a bleeding peptic ulcer (Arkkila et al., 2003)

More interestingly, prevalence of H pylori increases in gastric cancer patients (De Koster

et al., 1994) Hence, it is essential to study H pylori and its pathogenesis as it could

undoubtedly improve the understanding of these related gastroduodenal diseases and

refine their current treatments

2.4 Gastric mucosa and mucin

The human gastrointestinal tract is covered by a viscous and continuous layer of

mucus, which acts as a protective barrier for the underlying mucosa (Gendler & Spicer,

1995) The main component of mucus is a high molecular weight glycoprotein (mucin)

(Natomi et al., 1993) Interactions between bacterial pathogens and mucins derived from

human and animal species have been studied intensively In the case of H pylori, it is

most frequently found within and beneath the gastric mucous layer as well as attached to

the surface of gastric epithelial cell (Hazell et al., 1992; Falk et al., 1993; Olfat et al.,

2002) The organism is found in the duodenum only at sites of gastric metaplasia (Wyatt

et al., 1990) and is also discovered in colonizing patches of heterotropic gastric mucosa at

the upper esophagus (Borham et al., 1993), suggesting a specific tropism of the organism

for gastric mucosal surfaces in humans It is noted that few organisms exhibit such a strict

tissue tropism, which makes the adherence properties of H pylori to mucus layer

particularly interesting to examine

It has been reported that H pylori possesses a fibrillar hemagglutinin that

recognizes sialic acid containing structures on erythrocytes and on mouse adrenal cells

(Evans et al., 1988 & 1989) What has also been found is that mucins of different origin

such as porcine gastric mucin, bovine submaxillary mucin and human salivary mucin are

able to inhibit H pylori hemagglutination activity (Nakazawa et al., 1989; Piotrowski et

al., 1991) These observations indicate the likely existence of particular H pylori proteins

which could interact with sialic acid terminated molecules present on gastric epithelial

cells as well as in the gastric mucus layer

The role of interaction between H pylori and the gastric mucin in its pathogenesis is

yet to be established As has been suggested by other studies on bacterial attachment to

mucin such as E coli (Helander et al., 1997; Katayama et al., 1997), the attachment of H

pylori to gastric mucin might facilitate dissemination of the organism in the stomach

mucus layer and allow subsequent colonization on the underlying epithelium to occur

It is believed that the specific characteristics of mucins are affected by invading

microorganisms Human gastric mucin appears to be altered in patients with certain

disease such as gastritis or peptic ulcer (Younan et al., 1982) In addition, it is reported by

Hashiguchi (1993) that H pylori can enzymatically degrade mucin by specific protease

called mucinase thus alter its physicochemical properties Therefore, a detailed study on

the interaction of H pylori with several gastric mucin types will be useful for elucidating

the mechanism of adhesion

H pylori colonizes the gastric mucosa following its adherence and penetration of

the mucus layer covering the gastric epithelium (Hessey et al., 1990; Dunn et al., 1991)

Colonization of the gastric mucus layer protects the bacteria from extreme acidity of the

gastric tract and displacement from the stomach by forces such as those generated by

peristalsis and gastric emptying The clinical significance of the host-pathogen

interactions that allow the attachment of H pylori to human gastric cells remains to be

fully elucidated (Marshall, 1993) It is considered unlikely that chronic infection with H

pylori could occur in the absence of sophisticated adhesin-host cell interaction

2.5 Mucin binding proteins (MBPs) in bacteria

Interaction of H pylori with gastric mucin is not an isolated case (Mantle et al., 1989;

Lillehoj et al., 2004) A number of bacteria colonizing the mucosa can be found in the

mucus layer and adhere to the epithelial cells (Blomberg et al., 1993; Yoshiyama &

Nakazawa, 2000) Over the recent decade, several studies on the adhesion of lactobacilli

to epithelial cells and mucus have been reported (Rojas & Conway, 1996; Kirjavainen et

al., 1999; Edelman et al., 2002) showing that most lactobacilli used in probiotic products

adhere to human intestinal mucus In most cases, adhesion has been reported to be

mediated by proteins like collagen binding protein (CnBP) of Lactobacillus reuteri (Roos

& Jonsson, 2002) The binding activity of this protein is blocked by porcine intestinal

mucin and by a lectin with specificity for α-D-galactose, suggesting CnBP adheres to

mucin via a lectin-like interaction Roos & Jonsson (2002) characterized the adhesion of

Lactobacillus reuteri to intestinal mucus components in vitro Their results show that a

high molecular weight protein possessing the typical features of cell surface protein from

gram positive bacteria could adhere to components of mucus However, in the case of

Pseudomonas aeruginosa, it has been shown previously that the adhesion of the bacteria

to human respiratory mucins might be mediated by pilus and alginate but recently the

observation of the binding of non-pilated mutants to mucins and glycolipids suggested

the existence of a type of non-pilus adhesion (Nelson et al., 1990; Ramphal et al., 1991;

Plotwoski et al., 2001) In order to characterize the adhesion, Carnoy et al (1994)

examined the interaction of the outer membrane proteins from two adhesive P

aeruginosa strains with two types of glycoproteins secreted in human respiratory mucus,

which are mucin and lactotransferrin Several adhesins in both strains were identified

Non-typeable Haemophilus influenzae strains are the most common pathogens

encountered in patients with chronic bronchitis These organisms chronically colonize the

airways of patients and occasionally bind to mucus Quantitative studies of such adhesion

were reported by Vogel et al (1994), who developed a reproducible micro titer plate

assay to study mucin binding and found that 1hour of incubation is the most optimal for

binding assay The assay method has thus been established as a useful method for

studying bacteria-mucin interaction in vitro and a large number of bacterial proteins have

been reported possessing different levels of affinity with mucins of various origins

2.6 Family of peptidyl-arginine deiminase (PAD)

Peptidylarginine deiminase (PAD) is a family of enzymes that catalyze the

conversion of protein-bound arginine to citrulline.In mammalian cells, the enzyme is

mainly involved in post-translational modification of proteins that could have a big

impact on the structure and function of the target protein (Nissinen et al., 2003) Four

highly conserved isoforms of PAD enzymes have been identified in a wide range of

mammalian tissues (Terakawa et al., 1991) Both the enzymes and their products have

been widely studied with the knowledge that their close association with several human

diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS) have been

reported (Pritzker et al., 2000)

To date, the only prokaryotic PAD that has been purified is from bacterium

Porphyromonas gingivalis which has been associated with the initiation and progression

of adult-onset periodontitis It can convert both peptidyl-arginine and free L-arginine into

citrulline and produce ammonia concurrently in the presence of Calcium ions (McGraw

et al., 1999) and is believed to be a virulence factor of the organism by preventing acidic

cleansing cycles of the mouth

PADs in prokaryotic cells have been proven to be evolutionarily unrelated to

mammalian PADs as the two share low DNA sequence homology and possess different

sets of conserved enzymatic domains although catalyzing the same reaction However,

they are more closely linked with other prokaryotic enzymes such as arginine deiminase

and amidinotransferase by adopting a common fold and similar catalytic mechanisms

(Shirai et al., 2001)

2.7 Techniques used in protein functional studies

2.7.1 Western ligand blotting analysis

Western blot technique using labeled probes has been used widely to identify

potential protein receptors in bacteria by several investigators In 1998, a convenient

method for the detection of carbohydrate-binding activity of lectins was established by

Kamemura & Kato The method was mainly a combination of blotting of lectins onto

polyvinylidene-difluoride membranes, labeling by conjugated biotinylated probes and

horseradish peroxidase-streptavidin followed by detection using enhanced

chemiluminescence of enzyme reaction Using Western blot analysis, Calabi et al (2002)

identified two potential ligands of the Clostridium difficile surface layer proteins (SLP)

Mongodin et al (2002) investigated fibronectin binding protein (FnBPs) in

Staphylococcus aureus by Western ligand affinity blotting analysis Downer et al (2002)

used elastin as a ligand in Western blotting to identify elastin binding protein in S aureus

(EbpS) By affinity chromatography and Western blotting assays, Plotkowski et al (2001)

showed the interaction of different Pseudomonas aeruginosa outer-membrane proteins

(OMPs) with heparin In the light of the effectiveness of this method, Western blot

analysis was also employed for identifying the binding affinity of H pylori proteins with

biotin-labeled mucins in this study

2.7.2 Solid phase carbohydrate binding assay

Solid phase binding assay has served efficiently in identifying and screening the

proteins of interest in many bacteria (Kemeney & Challacomb, 1988) In this method, the

ligand is usually coated on solid phase micro titer plates and probe is added in order to

examine any possible interaction between the two Liang et al (1995) studied

vitronectin-binding activity in different fractions of Staphylococcus aureus using micro titer plates

with immobilized human vitronectin Van de Wetering et al (2001) used a solid phase

based binding assay to specify that Lipoteichoic acid (LTA) of Bacillus subtilis bound by

surfactant protein D (SP-D) instead of A (SP-A), of which both SP-D and SP-A have

been reported to be important for mediating binding of various gram-negative bacteria

that cause pneumonia The binding of uropathogenic Escherichia coli to host cells is

mediated at the tips of pili by the peptidoglycan (PepG) adhesin, which recognizes the

alpha (1-4) Gal disaccharide on the uroepithelial surface This phenomenon was

demonstrated by Mahmood et al (2000) using a specific solid phase assay In many other

studies of proteins and their ligands, solid phase assay is also successfully employed in

combination with western blot analysis for characterizing the mechanism of interaction

2.7.3 Immuno-gold labeled transmission electron microscopy (TEM) and

protein localization

With the development of antibody techniques and electron microscopy,

immuno-labeled electron microscopy has become an effective tool in displaying the

ultra-structural location of molecules in cells and tissues It is widely used in studying the

localization of proteins in eukaryotes and prokaryotes (Herrera, 1992) Immuno-labeled

transmission electron microscopy (TEM) is the most commonly used technique for

visualizing the exact location of molecules in cells Protein A-gold conjugates are

generally utilized as probes for immuno-staining as the gold particles can be readily

spotted under electron microscopy (EM)

Immuno-gold labeled TEM has long been used in the studies of protein localization

in H pylori In 1990, Hawtin et al used the monoclonal antibodies of urease to image the

localization of urease in H pylori Later, Drouet et al (1991) demonstrated the surface

localization of a 19kDa outer membrane protein and polysaccharides of H pylori The

localization of various proteins in H pylori have been determined by different

researchers using immuno-gold labeled TEM techniques and the method has been

credited as a powerful tool in studying the localization of H pylori proteins (Du & Ho,

2003), which excitingly provides important information on the understanding the

functions of these proteins in the bacteria

3.1 Culturing of H pylori

Stock culture of H pylori NCTC11637 was inoculated onto chocolate blood agar

plate consisting of blood agar base (Oxoid) with 5% horse blood The plates were then

incubated at 37°C in a humidified incubator (Forma Scientific) with 5% CO2 for three

days

3.2 Cloning of mbp (mucin binding protein) gene

3.2.1 Chromosomal DNA extraction

Genomic DNA of H pylori NCTC11637 was extracted according to the method

described by Hua et al (1999) Plate cultures of H pylori were harvested and transferred

to Eppendorf tubes containing 1.5 ml TE buffer (pH 8.0) The suspension was then

centrifuged at 8000 xg for 5 minutes at room temperature and washed once with TE

buffer An aliquot of 100 µl of 10 mg/ml Lysozyme (Sigma) was added to the bacterial

suspension and incubated at 37°C for 30 minutes to digest the cell wall before treating

with 100 µl of 10% sodium dodecyl sulfate (SDS) (Merck) for another 30 minutes at

37°C This was followed by treatment with 5 µl of 10 mg/ml Proteinase K (Boehringer

Mannheim Gmbh) at 56°C for 1 hour The DNA was subsequently extracted twice with

an equal volume of phenol (Sigma) and once with an equal volume of chloroform

(Sigma) The DNA was precipitated overnight with two volumes of absolute ethanol

(Sigma) and 20 µl of 3M sodium acetate (Sigma) at –20°C The precipitated DNA was

then washed with 70% ethanol The pellet was dried and resuspended in 50 µl of sterile

distilled water Two µl of 20 µg/ml RNase was added and the solution was incubated at

37°C for 20 minutes

to remove RNA The concentration of DNA was determined spectrophometerically at OD

260 nm

3.2.2 PCR amplification

Genomic DNA template from NCTC11637 was used for PCR amplification Fifty µl

of reaction mixture containing 0.2 mM of each deoxynucleotide, 1 unit Taq DNA

polymerase, 5 µl of 10X Taq polymerase buffer, 20 pmol each of forward and reverse

mbp primers and 50 ng of target DNA was prepared The PCR reaction was carried out in

a thermal cycler (Applied Biosystems Gene Amp PCR system 2400) The reaction

mixture was subjected to denaturation at 94˚C for 3 minutes, 30 cycles of reaction with

denaturing at 94°C for 1 minute, annealing at 62°C for 45 seconds and extending at 72°C

for 1 minute followed by final extension at 72°C for 10 minutes PCR products were

stored at –20°C and subsequently analyzed by agarose gel electrophoresis

The primers used to amplify the 993bp fragment encoding mbp by PCR were

designed based on the published sequence of H pylori 26695 strain (Tomb et al., 1997)

with the following sequence:

rMBP (Forward): 5-GCGAATTCCATATGAAAAGAATGTTAGCGGAGTTTG-3

rMBP (Reverse): 5-GACGGATCCTCAATAAAGTTGCATCGTTAC-3

PCR DNA samples mixed with 5X gel loading buffer were loaded into the wells of

the agarose gel containing ethidium bromide One kb DNA ladder (50 ng/µl) served as

molecular weight marker After electrophoresis at 100V in 0.5X TAE buffer, DNA bands

were visualized and photographed with filtered UV light

3.2.3 Restriction enzyme digestion and ligation into pET-16b

pET-16b plasmid was extracted using Plasmid Extraction kit (Qiagen) according to

the protocols provided by the manufacturer The concentration of the DNA was measured

using spectrophotometer (Nanodrop, Biofrontier) pET-16b plasmid and mbp from H

pylori genome were digested with NdeI overnight at 37°C followed by BamHI for 2

hours at 37°C Buffers compatible to both enzymes was used for the reaction The

products from the digestion were subsequently analyzed by agarose gel electrophoresis

and the desired DNA bands were extracted and purified using DNA extraction and

purification kit (Qiagen)

The concentrations of the purified mbp and pET-16b were measured, and the molar

ratio of the insert to the vector was varied from 3:1 to 7:1 to maximize ligation efficiency

An aliquot of 2 µl of T4 DNA ligase (3U/ml) (Promega) was used with 2 µl of 10X ligase

buffer in a 20 µl reaction mixture carried out at 4°C overnight After ligation, the mixture

was transformed into E coli TOP10 and screened on Luria-Bertani (LB) ampicillin plates

Ampicillin resistant clones were selected for further analysis

3.2.4 Transformation into BL-21 and selection of positive clones

A single colony of E coli BL21 (DE3) was inoculated into 10 ml of LB broth and

incubated at 37°C overnight with shaking at 120 rpm One ml of the culture was then

inoculated into 50 ml of fresh LB broth and incubated at 37°C for 3 hours at 250 rpm

Cultures were then transferred into sterile ice-cold polypropylene tubes and cooled in ice

for 10 minutes Cells were collected by centrifugation at 5000 xg for 10 minutes at 4°C

The pellet was resuspended in sterile ice cold 0.1M CaCl2 stored on ice for 30 minutes

and centrifuged again at 5000 xg for 10 minutes at 4°C The pellet was resuspended in 2

ml of sterile ice cold 0.1M CaCl2 with 50% glycerol, aliquoted and stored at –80°C until

use

Twenty µl ligation mixture was added to 200 µl of competent cells and mixed gently

The mixture was incubated on ice for 30 minutes and subsequently heat shocked at 42°C

for 90 seconds The cells were rapidly transferred onto ice and chilled for 2 minutes

Aliquot of 800µl of LB broth was added and incubated at 37°C for 45 minutes to allow

the bacteria to recover and express the plasmid After incubation, the cells were

centrifuged at 5000 xg for 5 minutes and the pellet was resuspended in 200 µl of fresh LB

broth and plated out on LB ampicillin agar plates

3.3 Expression and purification of recombinant mucin binding protein (rMBP)

3.3.1 IPTG-induced expression

A single colony of transformed E coli BL21 containing pET16b-mbp was

inoculated into 10 ml of LB ampicillin broth and incubated at 37°C with shaking at 120

rpm overnight Two ml of the culture was transferred into 50 ml of fresh LB Ampicillin

broth and incubated at 37°C with shaking at 250 rpm for 2 hours One ml of the culture

was then removed as an uninduced control IPTG (Biorad) at concentrations ranging from

0.1-0.4 mM was added for the induction of rMBP expression Aliquots of 1ml were

collected at hourly intervals for 5 hours during induction The cells were collected by

centrifugation at 12,000 xg for 5 minutes and the supernatant was discarded The pellet

was resuspended in 20 µl of PBS buffer (pH7.6) With 5 µl of 5X loading buffer added,

samples were boiled and examined by SDS-PAGE

The recombinant protein was run on SDS-PAGE electrophoresis under reducing

conditions of β-mercaptoethanol as described by Richard (2002) Loading buffer was

added to the different protein samples and boiled for 5 minutes to denature the proteins

Samples were loaded in a 4% stacking gel and resolved on a 10% separating gel using a

Mighty Small II SE 250 electrophoresis apparatus (Hoeffer Scientific Instruments) at

100V for 2 hours (Power Pac300, Biorad) Prestained precision marker (Biorad) was used

as molecular weight marker

After electrophoresis, SDS-PAGE gel was stained with Coomassie blue In brief,

the gel was immersed in staining solution containing Coomassie Blue R-250 for 2 hours

and then destained in destaining solution containing 10% acetic acid and 40% methanol

with several changes to obtain a clear background on the gel with well-defined protein

bands

3.3.2 Localization of rMBP in soluble and insoluble cell fractions

After induction with 0.1 mM IPTG for 3 hours, 1 ml of the culture was removed

and cells were collected by centrifugation and resuspended in 1 ml PBS buffer This cell

suspension was sonicated on ice 10 times for 10 seconds each with 10 seconds rest The

sonicate comprised the whole cell lysate (WCL) Twenty µl of WCL was preserved and

the remaining WCL was centrifuged at 12,000 xg for 15 minutes The supernatant served

as the soluble fraction and the pellet was regarded as the insoluble fraction The pellet

suspended in PBS buffer, the supernatant and the WCL were then subjected to

SDS-PAGE electrophoresis and visualized by Coomassie Blue staining

3.3.3 His-tag affinity purification

The E coli BL21 containing the recombinant plasmid was induced with 0.1 mM

IPTG for 3 hours and the cells were harvested by centrifugation at 12,000 xg for 5

minutes at 4°C The pellet harvested from 250 ml culture was resuspended in 20 ml 1x

binding buffer (Appendix) Protease inhibitor (Sigma) at a concentration of 5µg/ml was

added to the suspension Sonication was carried out on ice 10 times for 10 seconds each

with 10 seconds rest The suspension was then centrifuged at 12,000 xg for 15 minutes

and the pellet was resuspended in 10 ml 1x binding buffer and centrifuged again at

12,000 xg for 15 minutes The pellet was resuspended in 10 ml 1x binding buffer with

6M urea and incubated on ice overnight to completely dissolve the protein This

suspension was then centrifuged at 12,000 xg for 20 minutes and the supernatant was

filtered through a 0.45 µm micron membrane (Minisart) before being loaded onto the

Nickel-chelating column

As the target protein was mainly localized as inclusion bodies, purification was

carried out under denaturing condition The sample was loaded onto the column with 1x

binding buffer containing 6M urea and washed with 10 column volumes of the same

buffer Following this, the column was washed with 6 volumes of 1x washing buffer with

6M Urea The bound rMBP protein was then eluted using 6 volumes of 1x elute buffer

(Appendix) Fractions were collected during the purification and examined by

SDS-PAGE electrophoresis followed by Coomassie Blue staining The eluted protein was then

extensively dialyzed against PBS buffer (pH7.6) and concentrated using polyethylene

glycol 35000 (Merck)

Modified Biorad protein assay was used to determine the amount of protein in the

buffer A standard curve was prepared using different known concentrations of bovine

serum albumin (BSA) (Sigma) Determination of protein content was prepared by adding

10 µl of sample to 1 ml of Biorad protein dye solution (previously diluted 1:5 times) and

the OD was measured in a spectrophotometer (Spectronic Gennesys 5) at 595 nm

Concentration of purified rMBP protein was then calibrated using the known

concentrations of BSA

3.4 Protein identification by MALDI-TOF analysis

After SDS-PAGE gel electrophoresis, the bands of the purified target protein were

excised and subjected to trypsin in-gel digestion and purified with Zip-tip C18 Cartridge

column (Millipore) Sequence analysis and identification were carried out using

MALDI-TOF analysis at the Protein Proteomic Center, National University of Singapore

3.5 Detection of mucin binding capability

3.5.1 Western blot of Mucin I and II

Mucin was labeled with biotin before use A total of 2.04 mg of freshly prepared

biotin-NHS (Roche) dissolved in 100 µl DMSO was added to 24 mg of mucin dissolved

in 2 ml of PBS (pH 7.6) and incubated for 4 hours at room temperature with gentle

shaking The mixture was then dialyzed for 24 hours with 3 changes of PBS at 4°C

Labeled mucin was aliquoted and stored at –20°C until use Mucin Type I (Crude bovine

submaxilary gland mucin) (Sigma) and Mucin Type II (Crude porcine gastric mucin)

(Sigma) were used for the study

The SDS-PAGE gel run with purified rMBP was electrophoretically transferred

onto the Immobilon-P transfer membrane (Millipore) in a semi-dry Transblot cell

(Biorad) The transblotting was carried out at 0.15 mA for 1 hour After blotting, the

non-specific binding sites on the membrane were blocked by incubating in 5% skim milk in

PBS and shaken gently in a Belly Dancer for 1 hour The membrane was then washed

with PBS containing 0.05% Tween-20 (Merck) twice for 10 minutes each It was then

incubated with biotin labeled mucin (1:100 dilution in PBS containing 1% BSA)

overnight followed by 2 washes with PBS-T for 10 minutes each Streptavidin-POD was

then added (1:5000 dilution in 1% PBS-BSA) and incubated for 2 hours Finally, the

membrane was washed twice with PBS-T for 10 minutes and immersed in the substrate

solution (0.06g 4-chloronapthol in 20ml chilled methanol, 100 ml PBS and 60 µl H2O2)

until dark bands appeared Reaction was stopped with distilled water

3.5.2 Solid phase mucin binding assay at different pH and temperature

This procedure is a slight modification of the method used by Ascencio et al (1998)

A 96 well polystyrene micro titer plate (Nunc) was coated with 100 µl rMBP (100 ng per

well) and incubated overnight at 4°C Wells were aspirated and washed with PBS 2 times

at 3-minute interval each The non-specific binding sites in the well were blocked by

adding 100 µl 1% PBS-BSA per well and incubated at 4°C overnight The plates were

washed with PBS-T 3 times before adding 100 µl of biotin labeled mucin (1:100 dilution

in 1% PBS-BSA) to each well The plates were incubated at 37°C, 10% CO2 for 2 hours

followed by 3 washes with PBS-T Aliquot of 100 µl Streptavidin-POD (1:5000 dilution

in 1% PBS-BSA) was then added to each well and further incubated under the same

condition for 90 minutes Following this, the wells were washed 3 times with PBS-T and

twice with PBS before adding 100 µl substrate solution consisted of 0.02g

O-phenylenediamine hydrochloride (OPD) and 20 µl of H2O2 (Merck) in 25 ml substrate

buffer The reaction was allowed to continue at room temperature for 30 minutes Finally,

the reaction was stopped by adding 50 µl of 1M H2SO4 to each well and the binding was

quantified by measuring the color development in an ELISA reader (Magellan, Tecan) at

a wavelength of 492 nm

The solid phase mucin binding assay was performed under different pH conditions

Different pH was obtained by adjusting the pH of PBS-BSA buffer used for diluting

mucin, from pH 2.0 to 9.0 with 5M HCl The incubation with biotin labeled mucin was

carried out under the temperature range of 4-55°C Different reaction times were tested

by varying the incubation period with biotin labeled mucins from 0.5 to 3 hours

Different amounts of rMBP from 0.25-2 µg were used for the assay, and different

dilutions of mucins in 1%PBS-BSA (varying from 1:50 to 1:2500) were also tested in the

assay for optimizing the binding

3.5.3 Assay with mucins pretreated with neuraminidase and Na-metaperiodate

In order to further characterize the nature of binding between rMBP protein and

mucin, the biotin labeled mucins were pretreated with neuraminidase and

Na-metaperiodate, two commonly used agents for studying protein-lectin interactions by in

vitro assays (Gentsch & Pacitti, 1985) Neuraminidase is also termed as

Acetyl-neuraminyl hydrolase (Sialidase) that catalyzes the hydrolysis of N-acetyl-neuraminic

acid residues from glycoproteins and oligosaccharides (Gentsch & Pacitti, 1985)

Na-metaperiodate is a powerful oxidizing reagent catalyzing nonspecific oxidation of

carbohydrate groups on various molecules such as glycoproteins and glycolipids

(Marthieu et al., 1982)

The assay was tested using biotin labeled mucin treated with neuraminidase before

the reaction The pretreatment was carried out by incubating the biotin labeled mucin

with neuraminidase (Sigma) overnight at 37°C with gentle shaking The pretreatment of

biotin-labeled mucin with Na-metaperiodate was carried out by incubating the biotin

labeled mucin with Na-metaperiodate (Sigma) dissolved in PBS overnight at 37°C with

slow shaking

3.6 Detection of peptidyl-arginine deiminase (PAD) enzyme activity

3.6.1 Enzyme assay at different pH

The PAD activity was determined according to the method as described by McGraw

et al (1999), which was modified from the enzyme assay established for detection of

arginine deiminase activity (Monstadt & Holldorf, 1991)

The method was based on the amount of citrulline produced in the assay and the

concentration of citrulline was measured using the chemical colorimetric method (Boyde

& Rahmatullah, 1980; Smith et al., 1985) Standard curves were generated using free

L-citrulline (Sigma) with different known concentrations diluted with PBS Equal volume

of 0.5% diacetyl-monoxime (Sigma) was added to the solutions as a detecting reagent

The color development was quantified by the ELISA reader at 492nm The standard

curve was used for the calibration of citrulline concentration

The recombinant protein and H pylori WCL cell extract were incubated with 40 µl

of 5mM Benzoyl-Arg-ethyl-ester (BAEE) (Sigma) substrate buffer at 37°C for 1 hour

The reaction was stopped by the addition of 100 µl of stopping solution (Appendix) and

50 µl of 0.5% diacetyl-monoxime was added as a detecting reagent The mixture was

then heated at 100°C for 25 minutes and the color development was optically measured at

492 nm The 1% PBS-BSA was used as the negative control

The PAD assay was carried out over a pH range from 2.0 to 9.0 by adjusting the

pH value of substrate buffer Meanwhile, the enzyme assay was tested on substrate

solution containing free L-arginine instead of peptidyl-arginine Free L-arginine (Sigma)

was dissolved in PBS and then included in the substrate buffer with a final concentration

of 5 mM Different types of flavin nucleotides including flavin adenine mononucleotide

(FMN), flavin adenine dinucleotide (FAD) were added to the assays with a concentration

of 1µM as cofactors

3.6.2 Effect of PAD inhibitors on assay

Different concentrations of thiourea (Sigma) and L-cysteine (Sigma) dissolved in

distilled water at concentrations ranging from 1-20mM were added to the PAD assay

Their effects on the enzyme activity were compared with the assay in the absence of any

inhibitors

3.7 Antibody raising and purification

Antibody against rMBP was raised as described by Lo (2003) The study was

approved by the Animal Experimental Ethics Committee, National University of

Singapore Purified rMBP was injected into mice twice at a dosage of 20 µg at two weeks

interval Pre-immune blood was drawn to serve as a negative control Blood was drawn

two weeks after each injection Serum was separated from whole blood by centrifugation

and antibody titer was assayed by indirect ELISA method using 0.5 µg purified rMBP as

antigen

Protein A sepharose CL-4B (Pharmacia) affinity column was used to purify IgG

from antiserum The serum was diluted with 50 mM Tris-HCl buffer (pH7.0) and loaded

into the column The specific IgG was eluted with 0.1M Glycine-HCl buffer (pH3.0) and

neutralized with 1M Tris-HCl pH9.0 right after elution The specificity of antibody was

verified using Western blot analysis on cell extract of H pylori

In order to prepare the cell extract of H pylori, a 3-day-old culture of H pylori

grown on chocolate blood agar plate was harvested and resuspended in PBS buffer

Sonication of 10 seconds with 10 seconds rest for 6 times was applied to break the cells,

and the whole suspension served as the whole cell protein fraction Similarly, the cell

pellet of a 3-day old H pylori culture was lysed with 0.2M acid glycine (pH2.2) by gentle

stirring Supernatant collected was dialyzed against PBS at 4˚C overnight The acid

glycine extraction (AGE) comprised mainly cell membrane and membrane associated

proteins The protein concentrations were measured by Bradford assay and used for

Western blot analysis

3.8 Localization of MBP by immuno-gold labeled transmission electron microscopy

(TEM)

H pylori NCTC11637 cells harvested from a 3-day-old culture were washed with PBS

and fixed with 2% paraformaldehyde, 0.5% glutaraldehyde in PBS (pH7.4) for 1 hour at

4˚C, and then washed three times with PBS for 5 minutes each Dehydration was done by

incubating the cells in ascending graded series of ethanol at room temperature, including

5 min in 25%, 10 min in 50%, 20min in 75%, 20 min in 95% and 20 min in absolute

ethanol for 3 changes Infiltration into LR White (Merck) was carried out by passing the

sample through 1part ethanol and 1 part LR White for 30 min followed by passing

through 1 part ethanol and 3 part LR White for another 30 min It was then left in pure

LR White for two days with several changes The sample was then added into gelatin

capsule and polymerized at 50˚C until it was fully solidified, from which ultra-thin

sections were obtained using Microtome (Leica) The purified polyclonal mouse IgG

against rMBP (25µg/ml) was used as the primary antibody, while the secondary antibody

used was 20 nm gold –Protein A (1:20 diluted in PBS) (Sigma) The fixed sections of H

pylori cells at a thickness of around 90 µm were blocked with 0.1% PBS-BSA buffer and

then conjugated with the primary antibody at 37˚C for 2 hours They were then reacted

with the secondary antibody at 37˚C for an additional 2 hours The immuno-gold labeled

sections were further fixed in 2% glutaraldehyde at room temperature for 2 hours The

sections were then viewed under the Philips 208S electronic microscope The sections

labeled with either anti-rMBP IgG or immuno-gold were used as negative controls

3.9 Solid phase enzyme-linked immunobsorbent assay (SP-ELISA)

Detection of the immune response to rMBP in serum samples from patients with

different H pylori infection status was carried out according to the methods described by

Ng et al (2003)

A total of 100 serum samples which includes 50 from patients with peptic ulcer

diseases (PUD), and 50 from subjects with non-ulcer dyspepsia (NUD) were used for the

study PUD patients include those with duodenal ulcer or gastric ulcer NUD is defined as

the presence of symptoms thought to originate in the gastroduodenal region, in the

absence of any organic, systemic, or metabolic disease that is likely to explain the

symptoms (Tack et al., 2006) In short, NUD can be defined as patients with neither a

history of ulcer disease nor endoscopic evidence of ulcer disease All of the samples had

been tested for the presence of IgG antibodies against H pylori by ELISA and confirmed

as such by histology or culture methods

The positive control was pooled sera obtained from patients who showed strong

positive IgG titer against H pylori Dilutions ranged from 1:100 to 1:3200 were carried

out using the serum diluent (Appendix) for the positive control The negative serum used

was from the cord blood which showed negative IgG titer against H pylori This control

was diluted 1:100 using serum diluent rMBP was diluted with 0.5M carbonate buffer to

give a final concentration of 100 ng per well for use as antigen Aliquots of 100 µl were

then dispensed into 95 out of 96 wells of micro titer plates (Nunc) with one well left as

the negative blank control Coated plates were then incubated overnight at 4°C, aspirated

and blocked with 300 µl of serum diluent for another 24 hours of incubation at 4°C The

wells were then washed with PBS-T 3 times at 3-minute intervals Aliquots of 100 µl of

each test serum diluted 100 folds in serum diluent as well as the control serum were

added to the wells Each sample was carried out in triplicates

The plate was then incubated at room temperature for 90 minutes, aspirated and

washed 3 times with PBS-T Rabbit anti-human immunoglobulin labeled with horse

radish peroxidase (Dako) was used as the conjugate and diluted 1:50,000 with conjugate

diluent Aliquot of 100 µl of conjugate was added to each well and the plate was further

incubated for 90 minutes at room temperature After incubation, wells were washed 3

times with PBS-T and twice with PBS Then, 100 µl of substrate reagent containing 0.02

g OPD and 20 µl H2O2 dissolved in 20 ml of phosphate-citrate buffer was added to the

wells and the plate was incubated in the dark for 15 minutes at room temperature The

reaction was stopped by adding 50 µl of 2.5 M sulphuric acid and the plate was read

immediately using a micro titer plate reader (Magellan, Tecan) at a wavelength of 492

nm

4 1 Construction of pET16b-mbp expression vector

The mbp gene of was cloned into pET16b expression vector as shown in Figure

4.1A The 993 bp gene fragment of H pylori NCTC 11637 was amplified by PCR as

shown in Figure 4.1B The target gene fragment was inserted into pET16b expression

vector at multi cloning site of Nde1 and BamHI, which was fused at the downstream of

Histidine-tag for use in the over-expression of rMBP in E coli BL21

4.2 DNA and amino acid sequences of MBP

The amplified product of mbp was sequenced Complete sequence of mbp has a

length of 993 bp and encodes 330 amino acids Its G+C composition is 41.4% and its

deduced amino acid sequence is illustrated in Figure 4.2A The amplified product of mbp

of NCTC 11637 was sequenced and found to show >97 % identity to the published

sequences of HP0049 of H pylori 26695 (Tomb et al., 1997) and jhp0042 of H pylori

j99 (Alm et al., 1999) (Figure 4.2B) In order to minimize the inaccuracy in sequence

analysis of mbp, three rounds of independent PCR amplification and sequencing of the

mbp gene were carried out The deduced amino acid sequence of MBP of NCTC11637

showed 92.1% and 94.5% homology when compared with the deduced amino acid

sequences of HP0049 of H pylori 26695 and jhp0042 of H pylori J99, respectively

4.3 Expression and purification of rMBP

4.3.1 IPTG induced expression of rMBP in E coli BL21

The expression of rMBP was carried out by inducing E coli BL21 harbouring the

reconstructed pET16b-mbp with IPTG of concentrations ranging from 0.1-0.4 mM for up

to 5 hours However, increasing the IPTG concentration from 0.1-0.4 mM did not result

in further enhancement of rMBP expression as shown in Figure 4.3.1A The study shows

that the rMBP was found to reach its maximum expression after 3 hours of induction with

0.1 mM IPTG (Figure 4.3.1B) The expressed rMBP protein has a size of approximately

37 kDa that correlates with the predicted size of MBP based on the amino acid sequence

4.3.2 Localization of rMBP in cell fractions

The rMBP expressed by pET16b-mbp upon induction by 0.1 mM IPTG for 3 hours

was analyzed by SDS-PAGE electrophoresis The results show that the rMBP existed

mostly in the insoluble fraction probably as inclusion bodies (Figure 4.3.2) This is

further supported with the absence of protein band in the supernatant (soluble fraction)

Upon induction by IPTG, rMBP was expressed as shown in the induced WCL (Figure

4.3.2)

4.3.3 Purification of rMBP by His-tag affinity chromatography

Purification of rMBP from the insoluble fractions was carried out by His-tag affinity

chromatography under denatured conditions in ascending concentrations of imidazole

The protein profiles show that rMBP was successfully purified with elution buffer

containing 250mM imidazole (Figure 4.3.3) This confirms the over expression of rMBP

which was present mainly in the insoluble cell fraction of E coli BL21 The fractions

containing purified rMBP were pooled and dialyzed against PBS (pH7.6) and then

concentrated The His-tag affinity purification yielded around 20 mg of purified rMBP

for every 1 L culture of E coli BL21 pET16b-mbp

Figure 4.1 Construction and identification of mbp expression vector

A: Diagrammatic representation of the construction of pET16b-mbp mbp was cloned

downstream to the Histidine-Tag on pET16b; B: lane M, 1kb DNA Marker; Lane 1, reconstructed

vector (pET16b-mbp); Lane 2, amplified mbp fragment; Lane 3, pET16b digested with Nde1 and

BamHI

A

atgaaaagaa tgttagcgga gtttgaaaaa atccaagcga ttctaatggc gttcccccat

gagtttggcg actgggcgta ttgtatcaaa gaagccaggg gagctttttt aaacatcatt

caaaccatag ccaaacacgc tcaggtgcta gtgtgcgtcc acactaacga tattatcggt

tatgaaacgc ttaaaaactt acccggtgta gagatcgcaa ggattgacac taacgacaca

tgggctaggg attttggagc gatcagcgtt gaaaatcatg gcgttttaga gtgcttggat

tttggcttta atggctgggg gttaaaatac ccgtccaatt tagattatca agtgaatttc

aaactcaaaa gtttagggtt tttaaaacac cctttaaaaa cgatgcccta tattttagag

ggcgggagca tagaaagcga tggggctggg agcgttttaa ccaacaccca atgcctgtta

gaaaaaaatc gtaaccccca tttgaatcaa aatggaatag aaaacatgct taaaaaggaa

ttaggggcta aacaggtgct ttggtattct tatggctatc tcaaaggcga tgataccgat

agccataccg acacgctcgc tcgtttttta gataaagaca ccattgttta tagcacatgc

gaggatgaaa acgatgagca ctacacagcc ttaaaaaaaa tgcaagaaga attaaaaacc

tttaaaaaac tagacggaac gccctataaa ctcatccccc tagaaatccc caaagccatt

tttgatgaaa accaacaacg ctcgccggca acttatgtga attttttatt gtgcaataac

gctctcatcg tgcccactta caacgaccct aaagacgcgc tcattttaga aaccttgaaa

caacacacgc ccttagaagt gataggggtt gattgcaaca ccttaatcaa acagcatggg

agtttgcgtt gtgtaacgat gcaactttat tga