bglii thesis-protein purification

ISOLATION OF BglII RESTRICTION ENDONUCLEASE FROM

BACILLUS GLOBIGII

BY NADIPINENI ASHOK KUMAR

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Isolation of BglII Restriction Endonuclease From Bacillus globigi....................................11 Abstract........................................................................................................................62 Introduction..................................................................................................................7

2.1 BglII Restriction modification System:...............................................................83 Review of literature:..................................................................................................11

3.1 Genes, Proteins and their structures:.................................................................113.2 Protein Purification:...........................................................................................133.3 Principles of Chromatographic separation:.......................................................153.4 Size Exclusion Chromatography:......................................................................163.5 Adsorption Chromatography:............................................................................18

3.5.1 Separation Techniques in Adsorption Chromatography:..........................183.6 Methods for the analysis of Protein Size, Quantitation and molecular weight determination:................................................................................................................27

3.6.1 Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)28

3.6.2 Lab-on-a-chip technology..........................................................................303.6.3 Size exclusion chromatography (SEC)......................................................303.6.4 Mass spectrometry.....................................................................................31

3.7 Restriction Endonucleases:................................................................................343.7.1 MECHANISM OF ACTION:....................................................................353.7.2 DIVALENT CATIONS:............................................................................36

3.8 Agarose Gel Electrophoresis:............................................................................363.8.1 ELECTROPHRORESIS UNIT:................................................................373.8.2 AGAROSE:...............................................................................................373.8.3 ELECTROPHORESIS BUFFER:.............................................................383.8.4 LOADING DYE:.......................................................................................383.8.5 ETHIDIUM BROMIDE:...........................................................................38

4 Materials and Methods..............................................................................................394.1 Materials:...........................................................................................................39

4.1.1 BACTERIAL STRAIN:............................................................................394.1.2 BACTERIAL MEDIA AND AGAR PLATES.........................................394.1.3 ENZYMES:...............................................................................................394.1.4 CHEMICALS:...........................................................................................394.1.5 PREPARATION OF STOCKS SOLUTIONS:.........................................394.1.6 BUFFERS:.................................................................................................414.1.7 EQUIPMENT:...........................................................................................43

4.2 METHODS:.......................................................................................................444.2.1 Genomic DNA isolation:...........................................................................444.2.2 ISOLATION OF PLASMID USING MINI PREP KIT:...........................444.2.3 ISOLATION OF RESTRICTION ENDONUCLEASE............................454.2.4 RESTRICTION DIGESTION:..................................................................474.2.5 SDS-PAGE:...............................................................................................484.2.6 SILVER STAING METHOD:..................................................................494.2.7 AGAROSE GEL ELECTROPHORESIS:.................................................50

5 Results........................................................................................................................51

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5.1 Purification of BglII Restriction endonuclease by ion-exchange chromatography (batch method):...................................................................................51

5.1.1 DEAE-Cellulose........................................................................................515.1.2 CM-Cellulose.............................................................................................525.1.3 Optimization of salt gradient for the elution of BglII:...............................55

5.2 Isolation of BglII using the optimized DEAE-cellulose chromatography:.......576 Discussion..................................................................................................................597 Conclusion.................................................................................................................618 References..................................................................................................................62

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FiguresFigure 1a BglI recognition sequence 7Figure 1b BglII recognition sequence 7Figure 2 BglII amino acid sequence 8Figure 3 Size exclusion

chromatography15

Figure 4 Ion exchange chromatography

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Figure 5 Affinity chromatography 19Figure 6 Restriction digestion

mechanism33

Figure 7 Restriction digestion and phosphodiester bond breakage

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Figure 8 Electrophoresis Power Pack and Gel Tank

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Figure 9 Agarose structure 36Figure 10 Ethedium bromide staining 36Figure 11 Purification of BglII using

anion exchange chromatography (pH 5.6 and 8.0)

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Figure 12 Purification of BglII using cation exchange chromatography (pH 2.8 and 7.2)

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Figure 13 SDS-PAGE on purified samples

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Figure 14 Optimization of elutions using salt gradient -1h

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Figure 15 Optimization of elutions using salt gradient – overnight

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Figure 16 Purification of BglII from 100ml culture using DEAE chromatographic method

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Tables

Table 1 Genetic code 10Table 2 Classification of restriction

endonucleases32

Table 3 Restriction digestion setup 45Table 4 Preparation of gel loading

samples46

Table 5 Resolving gel mix 46Table 6 Stacking gel mix 47

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1 ABSTRACT

Type II restriction endonuleases are defined as double stranded nucleases that recognize

specific DNA sequence and cleave at a defined point within or close to that sequence.

They require only Mg++ as a cofactor. These are the basic tools in genetic engineering

and molecular biology. These endonucleases are often found in prokaryotes. Their

primary function is defense against foreign organisms such as viruses. The present study

was aimed to isolate BglII restriction endonuclease from its native source (Bacilllus

globigi). BglII was isolated using ion-exchange chromatography techniques. The isolated

enzyme was found to have higher activity then commercial enzyme. The isolated BglII

enzyme showed Type II restriction endonuclease activity which was demonstrated by

digesting a know plasmid (pCI-mammalian expression vector) which carries a single

BglII site.

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2 INTRODUCTION

The field of recombinant DNA and genetic engineering relies on enzymes and techniques

that permit the specific cutting, splicing and sequencing of DNA molecules the

recognition of the recombinant products and the introduction of recombinant molecules

into almost any kind of cell. The filed has become one of the most vigorous in science,

owing to the analytical power it gives to study gene structure and expression, to the

ability it gives us to make many biological products in lower organism, and the

possibility of directly changing the genotype of organisms of commercial interest. The

study of gene themselves, as opposed to their effects, become possible with the advent of

Restriction Enzymes.

Restriction enzymes bind specifically to cleave double stranded DNA at specific sites or

a particular sequence known as recognition sequence. These enzymes have been

classified into three groups; Type I, Type II and Type III restriction enzymes (Yawn

1981). Type I and Type III enzymes carry modification (methylation) and ATP

dependent restriction (cleavage) activities in the same protein. Type III enzymes cut the

DNA at recognition site and then dissociate form the substrate (Pickarowiez et al 1976).

Neither Type I nor Type III restriction enzymes are widely used in molecular cloning.

Type II restriction/modification systems are binary systems consisting of a restriction

endonuclease that cleaves at specific sequences of nucleotide and a separate methylase

that modifies the same recognition sequence. Thus the presence of a restriction enzyme

and modification of a DNA by methylation are coupled for every endonuclease.

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Type II enzymes are structurally simple protein and cut within or very close to

recognition site in double stranded DNA. Recognition sequences often has dyed axis of

symmetry ranging 4-8 nucleotides in length. Several enzymes display recognition

specificities termed as relaxed. These enzymes can not distinguish between purine or

pyrimidine bases at certain position within their recognition sequence. Several enzymes

isolated from different sources recognize the same sequence. They can be functionally

distinguished from each other by the site of cleavage, site of methylation and sensitivity

of cleavage reaction to DNA methylation and designated as isoschizomers. A large

number of these enzymes, isolated from bacterial species are known to be used to analyze

and manipulate DNA of plasmids, phage, bacteria and eukaryotic genomes. On the basis

of theoretical consideration it can be postulated that there are many more potential sites

of which specific cleaving enzymes must exit in the vast micro flora yet to be explored.

2.1 BglII RESTRICTION MODIFICATION SYSTEM:

Bacillus Sutilis (B.globigi) restriction modification system consists of an BglI and BglII-

endonuclease and BglI and BglII- methylase which recognizes and modify the

hexanucleotide sequence (5’ GCCNNNNNGGC 3’ and 5’ AGATCT 3’). The BglI

catalyzes the hydrolysis of phosphodiester bond in the recognition sequence and result in

in 3’ overhang (Fig 1a). Where as BglII catalyze the hydrolysis fo phsophodiester bond in

the recognition sequence and result in 5’ overhang (Fig 1b).

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5’ GCCNNNNNGGC 3’ 3’ CGGNNNNNCCG 5’

5’ GCCNNNN 3’ 5’ NGGC 3’ 3’ CGGN 5’ 3’ NNNNCCG 5’

Figure 1a: BglI endonuclease recognizes 11 base pair sequence and hydrolyzes the phosphodiester bonds between 4th N and 5th N on either strands from the 5’ end. This results in 3’ overhang.

5’ AGATCT 3’3’ TCTAGA 5’

5’ A 3’ 5’ GATCT 3’ 3’ TCTAG 5’ 3’ A 5’

Figure 1b: BglII endonuclease recognizes hexa nucleotide sequence and hydrolyzes the phosphodiester bonds between AG from the 5’ end. This results in 5’ overhang.

BglI and BglII methylases which are part of the BglI and BglII restriction modification

system catalyzes the methylation of the recognition sequences and protect host related

DNA from BglI and BglII restriction endonuclease. This project is aimed at selective

isolation of BglII restriction endonuclease from Bacillus globigi. BglII restriction

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BglI

BglII

endonuclease is of 223 amino acids in length (Fig 2) and weights about 25.7 KDa. The

theoretical pI of BglII is 6.41.

10 20 30 40 50 60 MKIDITDYNH ADEILNPQLW KEIEETLLKM PLHVKASDQA SKVGSLIFDP VGTNQYIKDE

70 80 90 100 110 120 LVPKHWKNNI PIPKRFDFLG TDIDFGKRDT LVEVQFSNYP FLLNNTVRSE LFHKSNMDID

130 140 150 160 170 180 EEGMKVAIII TKGHMFPASN SSLYYEQAQN QLNSLAEYNV FDVPIRLVGL IEDFETDIDI

190 200 210 220 VSTTYADKRY SRTITKRDTV KGKVIDTNTP NTRRRKRGTI VTY

Figure 2: BlgII amino acid sequence (Ref: PubMed)

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3 REVIEW OF LITERATURE:

3.1 GENES, PROTEINS AND THEIR STRUCTURES:Proteins carry out most of the active processes of the cell. They are the active machinery

that delivers the function encoded in the genes. The central dogma of life describes the

flow of information form DNA to protein. Transcription is the first step, in which the

original information of the DNA gene is copied into the blueprint molecule called

mRNA. In the translation step, the coding sequence of the mRNA is used as template to

build the amino acid sequence in the protein polymer chain. A protein complex called

RNA polymerase performs transcription whereas translation occurs at the ribosome, a

large rRNA-protein complex. The DNA polymer is built up by four different monomers,

called nucleotides, and their linear combination can be experimentally determined by

DNA sequencing. The protein polymer is built up from 20 different amino acid

monomers and the genetic code (Table-1) tells us how the gene sequence is translated

into the amino acid sequence of the protein.

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T C A G

T

TTT Phe (F)TTC Phe (F)TTA Leu (L)TTG Leu (L)

TCT Ser (S)TCC Ser (S)TCA Ser (S)TCG Ser (S)

TAT Tyr (Y)TAC TAA STOPTAG STOP

TGT Cys (C)TGC TGA STOPTGG Trp (W)

C

CTT Leu (L)CTC Leu (L)CTA Leu (L)CTG Leu (L)

CCT Pro (P)CCC Pro (P)CCA Pro (P)CCG Pro (P)

CAT His (H)CAC His (H)CAA Gln (Q)CAG Gln (Q)

CGT Arg (R)CGC Arg (R)CGA Arg (R)CGG Arg (R)

A

ATT Ile (I)ATC Ile (I)ATA Ile (I)ATG Met (M) START

ACT Thr (T)ACC Thr (T)ACA Thr (T)ACG Thr (T)

AAT Asn (N)AAC Asn (N) AAA Lys (K)AAG Lys (K)

AGT Ser (S)AGC Ser (S)AGA Arg (R)AGG Arg (R)

G

GTT Val (V)GTC Val (V)GTA Val (V)GTG Val (V)

GCT Ala (A)GCC Ala (A)GCA Ala (A)GCG Ala (A)

GAT Asp (D)GAC Asp (D)GAA Glu (E)GAG Glu (E)

GGT Gly (G)GGC Gly (G)GGA Gly (G)GGG Gly (G)

Table 1: The genetic code. Nucleotides are shown by their single letter code and amino acids by their three letter code. The nucleotide sequence is read in triplets, called codons. The translation machinery recognizes the codons on the mRNA and incorporates the corresponding amino acid to the peptide amino acid to the peptide chain. The genetic code is highly conserved among species, but the relative frequencies at which the codons occur in the genes vary considerably.

The amino acid sequence uniquely identifies individual proteins. But the function of

proteins depends critically on how the linear chain of amino acids folds into the three-

dimensional structure of the protein. DNA in the cell almost always forms the structure of

an elongated double stranded helix, independently of the sequence. Proteins on the other

hand, are very diverse in their structures. The amino acid sequence is called the primary

structure. Short-range interactions favor the formation of low energy conformations

called secondary structures elements are the spiral -helix and the elongated β-strand.

Secondary structure elements are connected by flexible loops or unstructured elements

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called random coils. The spatial conformations of the different secondary structure

elements define the structure of the proteins and are formally referred to as tertiary

structure.

Based on the gene sequence it is possible to predict the primary structure of the encoded

protein using the genetic code (Table 1). The prediction of secondary structure is not as

reliable but can be made based on the amino acid sequence, as the amino acids have

different properties to form secondary structure elements. Specific combinations of

different amino acids favor the formation of -helices, β-strands, or random coils,

although other factors also contribute. Thus in rare cases, an identical amino acid

sequence can be helical in one context but β-strand in another. It is currently not possible

to predict the structure of proteins based on their sequence alone. However, one powerful

way to enable prediction of both structure and function is to make comparisons to closely

related sequences. Proteins that are homologous, i.e. they have evolved from a common

ancestral protein; retain a similar function, structure and usually also a high degree of

sequence identity. For closely related proteins it is usually possible to correct attribute

homology based on sequence alone. An empirically derived threshold from sequences

sharing identities above 30%, or sometime lower if several related sequences are known,

have been defined for attributing homology based on sequence.

3.2 PROTEIN PURIFICATION:The set of complex isolation and purification steps involved in the recovery of the

product is generally referred to as downstream processing. In the design of the process

leading to product recovery the nature of the starting material is a key parameter. Equally

important, however is the desired quality of the final product, i.e., the maximum

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acceptable level and the chemical nature of impurities or contaminants. The demands

regarding purity will be highest for human therapeutics, somewhat less for a number of

diagnostics and often considerably lower for industrial enzymes. Regulatory issues and

environmental considerations (GMP, various national laws, etc.) are also important,

especially in the case of industrial down stream processing. The design and validation of

standard operating procedures (SOP), between batch in-place cleaning (CIP) and

sanitizing (SIP) procedures or the validation of operational parameters such as column

leeching and the removal of certain impurities (endotoxins, virus DNA, etc.) have to be

taken into account. [1]

A typical recovery process can be roughly subdivided into four steps [2]. First, the

product is separated from the producing organism and other insolubles by a solid//liquid

separation step, such as centrifugation or filtration. This may required cell rupture in the

case of intracellularly enriched substances or resolubilization in the case of inclusion

bodies [3]. In the second isolation step, substances that differ considerably in their

physico-chemical character are removed from the product. The methods used are either

highly specific and based on bioaffinity interactions or nonspecific such as salting out,

solvent extraction or batch adsorption. If well designed this step should result in a

considerable increase in product concentration to facilitate the following purification

steps. These usually employ highly selective methods to remove from the product

substances having similar physical properties and biochemical functions. At this point,

chromatography plays a predominant role as a recent analysis of more than a hundred

laboratory-scale protein purification has demonstrated [4]. The fourth step is polishing

and may include gel filtration, crystallization and lyophilization of the final product.

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Chromatography has been the primary preparative separation method in biology and

biochemistry since 1906, when the Italo-Russian botanist Tswett separated the pigments

of chlorophyll by passing petroleum ether extracts through a column packed with

powdered chalk [5]. In the 1930s, column chromatography become an important tool for

the separation of natural products in general, while the introduction of paper

chromatography pioneered the use of chromatographic separation techniques for the

microanalysis of biological samples. Various characteristics of biomolecules ranging

from general physico-chemical properties to biospecific interactions have been exploited

for their chromatographic separations. Although several modes of chromatography have

been recognized, most effort has been devoted to the development of (linear) elution

chromatography. The development of high performance liquid chromatography (HPLC)

in the 1970’s allowed high speed analysis and set new standards of precision and

resolution in the liquid chromatography of small molecules. In the 1980’s the main

feature of HPLC (i.e., high resolution, short analysis time and high sensitivity) have been

applied to the preparative separation of fast analysis of proteins and other biopolymers.

3.3 PRINCIPLES OF CHROMATOGRAPHIC SEPARATION:

Chromatographic separations are traditionally semi-batch procedures. The sample

mixture is introduced at one end of a column packed with the stationary phase through

which the mobile phase is passed. The separation of the sample components takes places

along the axis of the column due to differences in the molecular dimensions or due to

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hydrophobic, ionic, or biospecific interactions between the sample molecules and the

stationary phase. On the production scale, a continuous separation process would be

preferable, as such processes are usually more economical, can be automated more easily,

and achieve a more standardized product quality. Furthermore, they enable a better

utilization of the adsorbant and mobile phase and may facilitate recycling. The

advantages of continuous chromatographic separations are fully realized only in large

scale processes; therefore such chromatographic processes are found mainly in the

petrochemical and sugar industries.

3.4 SIZE EXCLUSION CHROMATOGRAPHY:In size-exclusion chromatography (SEC), also called gel filtration or gel permeation

chromatography (GPC), the sample molecules are separated according to their

hydrodynamic diameter [5]. The sample is passed through a column packed with an inert

porous material that possesses appropriate pore size distribution and volume. Separation

occurs due to differences in the intraparticulate void volume explored by the sample

components of different molecular dimensions. Molecules larger than the upper exclusion

limit cannot enter the intraparticulate void space, whereas sufficiently small molecules

have access to all the pores. For sample components of intermediate molecular

dimensions, the retention volume, Vr, is given by:

Vr = Vo + KDVi (1)

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where Vo is the interstitial volume, KD is the distribution ratio and Vi is the

intraparticulate void volume.

SEC is commonly practiced with crosslinked dextran such as Sephadex, modified agarose

and polyacrylamide-based gels that do not generally permit the use of high pressures.

However, column packings of high mechanical stability, ranging from silica-based

materials to macroreticular rigid polymers, are increasingly employed in SEC-HPLC.

Figure 3: Size Exclusion Chromatography [6]

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3.5 ADSORPTION CHROMATOGRAPHY:

Adsorption chromatography is based on the different distribution of each substance

between the stationary and the mobile phase due to interactions between the components

and the chromatographic surface.

3.5.1 SEPARATION TECHNIQUES IN ADSORPTION CHROMATOGRAPHY:

Liquid chromatography is the most important purification method for biological

substances when high resolution is required. Its versatility and flexibility is unsurpassed.

The molecular qualities most commonly exploited in separations are size, ionic and

hydrophobic properties as well as certain biospecific interactions.

3.5.1.1 ION-EXCHANGE CHROMATOGRAPHY

Ion-exchange chromatography (IEC) has been the most widely applied technique in

preparative protein chromatography both in the laboratory and on a production scale [7].

The recovery of biological activity is usually excellent. Weak anion and cation

exchangers carrying diethylaminoethyl (DEAE) and carboxymethyl (CM) ligates,

respectively, are most often used in both conventional and high performance IEC. Strong

ion-exchangers with sulfonic acid or quarternary ammonium groups are also widely used

in protein purification. In IEC, the sample components are retained by virtue of

electrostatic interactions between the charged eluate molecules and the oppositely

charged chromatographic surface. Consequently retention occurs on ionexchangers when

the sign of the fixed charges at the surface is the opposite of that of the net charge of the

protein. However, this is only a rule of thumb, as the relationship between retention and

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net charge is usually not so straightforward. This is because the charge distribution over

the surface of the protein molecule is not uniform and because steric effects also play an

important role in determining the magnitude of the interaction. Two models currently

used in protein IEC (the stochiometric displacement and the electrostatic interaction

model) link the respective retention factors to the ionic strength of the mobile phase and

the number of charged groups involved in the adsorption/desorption process

[8-11].

Figure 4: Ion Exchange Chromatography [12]

Gradient elution with increasing salt concentration is most widely used in the IEC of

proteins. When the salt concentration of the eluent is increased, charges present on the

protein molecules and at the surface of the stationary phase are screened and as a result

attraction between the protein molecules and the stationary phase is diminished. Since the

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net charge of the protein and, in the case of the weak ion-exchangers, the charge of the

chromatographic surface are both pH-dependent, control of the mobile phase pH is very

important in IEC and great attention has to be paid to the nature of the buffer as well. A

pH gradient may also be used for protein elution; however, due to the technical

difficulties in generating smooth and reproducible pH gradients, they are less commonly

employed than salt gradients.

3.5.1.2 AFFINITY CHROMATOGRAPHY

Many biological processes involve highly specific interactions based on molecular

recognition. The unique biospecificity arises from a synergistic effect of combined van

der Waals, electrostatic, hydrophobic and hydrogen-bonding interactions, the effect of the

aqueous medium and the complementary steric arrangement of the interacting moieties.

Affinity chromatography, a term introduce by Cuatrecasas et al. in 1968 [13], exploits

such biospecific interactions for separation purposes. The technique was first practiced in

the form of traditional column chromatography during the 1970's. Later, it was adapted to

HPLC, combining the high selectivity characteristic of biospecific interactions with the

speed, efficiency, and other features of HPLC. It has rapidly developed into a fast, highly

selective method for separating a wide variety of complex biological molecules, as well

as viruses and cells. In production-scale chromatography, affinity-based separations are

often seen as single-step alternatives to the multi-step processes that incorporate IEC,

HIC/RPC and SEC separations [14,15].

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Figure 5: Affinity Chromatography [16]

The surface of an affinity sorbent must be highly hydrophilic without functions that

would elicit nonspecific interactions [17]. The most commonly used supports are based

on agarose, porous glass, silica, polyacrylamide, methacrylate and cellulose [18]. Fibrous

supports were developed specifically for preparative applications [19]. A biospecific

ligand may be covalently linked to the stationary phase surface via a hydroxyl, amino, or

carboxyl function. Frequently, a spacer arm is used to anchor the ligand to the support

surface. A variety of preactivated stationary phases are commercially available to which

the affinity ligands, e.g. antibodies, antigens, lectins, receptors, enzyme inhibitors,

hormons or biomimetic ligands, may be attached using standard immobilization

21

techniques. While highly specific binding is the essence of affinity chromatography, the

binding between the affinant and the product to be isolated should not be too strong.

Otherwise desorption and column regeneration may require harsh conditions with

concomitant denaturation of the product or even the affinant itself.

Protein A and Protein G are widely used for IgG isolation, even though they exhibit some

subclass specificity [20-22]. Recent advances in genetic engineering have also helped to

expand the scope of affinity chromatography. It is possible to fuse an affinity tag such as

a Protein A [23] or glutathion-S-transferase [24] sequence to the recombinant protein,

which increases the product affinity for an immunoglobulin or glutathione column

considerably an allows selective removal from most contaminants.

For product isolation, affnity chromatography is performed in the frontal mode under

conditions where only the product binds to the stationary phase, while all other feed

components move through the bed unretained. Nonspecific adsorption can be reduced by

carefully choosing the operating conditions, i.e. the pH and salt concentration of the

mobile phase and the additives used.

3.5.1.3 METAL AFFINITY CHROMATOGRAPHY

Extending Helfferich's concept of ligand exchange to the separation of biomolecules,

stationary phases with immobilized metal ions have been used in the chromatography of

proteins and nucleotides and to investigate the surface topography of protein histidine

residues [25, 26]. The technique, called immobilized metal affinity chromatography

(IMAC) or metal interaction chromatography (MIC), has even gained significance in

large-scale protein purification. IMAC is based on the interaction between a metal-ion

22

electron acceptor (Lewis acid) and an electron donor (Lewis base) on the surface of a

protein. With a few exceptions, such as the interaction of phosphoproteins with Fe 3§

ions, proteins interact through their surface histidine and - to a lesser extent - their

tryptophane residues. Metal ions of intermediate polarizability such as Cu 2 +, Ni 2§ Zn 2

+, and Co 2§ are particularly suited for interaction with proteins as they may interact not

only with the nitrogen in amino- and imino-groups, but also with oxygen and sulfur.

Metal ions are immobilized on the stationary phase by chelating

functions, such as the two-dentate ligand IDA (iminodiacetic acid) bond to the support.

The nature of the chelating agent is of consequence. If the immobilization of the metal

ion involves several coordination sites, the metal is bound strongly to the

chromatographic surface and bleeding is less likely to occur. At the same time, however,

the number of coordination sites available for proteis binding by the stationary phase is

also reduced. Traditionally, soft agarose beads were used for protein purification by

IMAC. Since then a number of rigid supports and membrane adsorbers suitable for HPLC

applications including IMAC. have become available [27,28]. The mobile phase in IMAC

is a buffered salt solution and the strength of the metal-protein interaction is modulated

by the type and concentration of the salt [27]. Generally, retention of acidic proteins

decreases, whereas the retention of basic proteins first increases, then decreases with

Chromotography in the Downstream Processing of Biotechnological Products 43

increasing ionic strength. In preparative IMAC, the separation is achieved by

differential elution with stepwise changes in the salt concentration. The pH also

influences the retention behavior of proteins. Elution in IMAC most commonly involves

lowering the pH of the mobile phase to 6 so that the histidine residues are protonated. A

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mild competing agent such as glycine, histidine, or imidazole, or an organic modifier is

also frequently used for the elution of the protein. Complexation of the metal ions by

EDTA or the use of etal ions (which compete for the binding sites on the protein) in the

mobile hase are further means for protein elution. In IMAC, the three-dimensional

protein structure is not strongly affected by hromatographic surface binding, therefore the

biological activity of the products usually well preserved. In order to facilitate the

isolation of recombinant proteins by IMAC, an ligohistidine tag can be added, thus

imparting to them a strong ffinity towards Cu 2 § This approach may have advantages

over the use of arger tags such as Protein A, which may be difficult to remove in order to

obtain the intact protein product.

3.5.1.4 HYDROPHOBIC INTERACTION CHROMATOGRAPHY

Hydrophobic interaction chromatography (HIC) was developed in the 1970s specially for

the separation of proteins using agarose-based stationary phases with a low density of

mildly hydrophobic ligands [29,30]. However, most of these early stationary phases

contained ionic groups as well as hydrophobic groups, thus engendering a mixed

retention mechanism. More recently, rigid macroporous silica or polymeric supports have

been introduced that are covered with a covalently bound hydrophilic surface layer which

incorporates appropriate hydrophobic ligands, such as short alkyl, aryl or polyether

chains at comparatively low concentration [31]. Protein retention and selectivity depend

on the nature and size of the hydrophobic moieties. Retention s enhanced by high salt

concentration in the aqueous mobile phase and therefore gradient elution with decreasing

salt concentration is most commonly used in the HIC of proteins. Due to these

comparatively mild operating conditions, the molecular integrity of the native protein is

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normally preserved and no significant loss of biological activity occurs. For this reason,

HIC is widely employed in the preparative and process-scale isolation and purification of

proteins [32]. It should be noted, however, that some slight changes in the protein

structure may occur in HIC due to a weakening of the hydrophobic forces responsible for

maintaining that structure [33].

3.5.1.5 REVERSED-PHASE CHROMATOGRAPHY

Reversed-phase chromatography (RPC) was introduced in 1950 for the separation of

nonpolar substances [34]. It has become the predominant branch of analytical HPLC also

in the life sciences and in biotechnology, so that, to date, an estimated 60%-70% of all

HPLC separations are carried out by RPC. Most commonly, bonded high performance

stationary phases prepared by covalently binding hydrophobic ligands such as C4-, C8-,

Cxs-alkyl chains or aromatic functions to the surface of a rigid siliceous or polymeric

support are used [35]. Due to the strong hydrophobic character of the stationary phase,

proteins and peptides are bound very strongly from an undiluted aqueous mobile phase so

that their elution requires the use of hydro-organic eluents. The separation of peptides and

protein in RPC is typically carried out by gradient elution with increasing concentration

of an organic modifier such as acetonitrile, methanol, tetrahydrofuran and isopropanol. In

addition, the mobile phase usually contains low levels of trifluoroacetic or phosphoric

acids. The use of RPC in preparative work requires the refolding of the product into its

native configuration after separation. This has been shown to be possible for a number of

peptides and smaller proteins which are of interest to the pharmaceutical industry, such as

h-insulin, h-growth hormone (hGH), tissue plasminogen activator (TPA) and various

interleukins [36].

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3.5.1.6 HYDROXYAPATITE CHROMATOGRAPHY

Hydroxyapatite (HA) has been used as the stationary phase for biopolymer

chromatography since 1956 [37,38]. While these early materials were soft powders

(Tiselius apatite), new production procedures were developed in the early 1980s, which

resulted in HPLC compatible beads [39]. Recently, fluoroapatite (FA), which has similar

properties to HA but significantly higher mechanical strength and chemical stability, has

become available. Today, HA columns are commercially available for both analytical and

preparative purposes from different suppliers. HA and FA will bind both negatively and

positively charged substances, yet a simple ion-exchange mechanism does not account

for the observed chromatographic behavior. Both apatite compounds only bind proteins

which possess an intact three-dimensional structure; therefore denaturation of a protein

by chaotropic substances or by heat, largely prevents it from being adsorbed on apatite.

The physico-chemical phenomena underlying the separation were examined in the 1970's

and several retention models have been proposed [40]. Since chromatography on FA

gives similar results to chromatography on HA, it was concluded that neither the hydroxy

nor the fluoro groups play a dominant role in the separation. Two types of binding sites

are thus present at the chromatographic surface: the calcium ions (C-sites) and the

phosphate groups (P-sites) which interact with the amino and carboxyl groups of the

protein. In contact with a mobile phase of neutral pH and above, the apatite surface

carries a net negative charge, as a result of a surplus of phosphate groups. This is

amplified in most chromatographic separations on apatite by the use of a phosphate

buffer as the mobile phase. Positively charged proteins bind by electrostatic interactions

26

to the negatively charged surface. Desorption is brought about by a high concentration of

anions, such as F-, Ct- and SCN-, in the mobile phase. By a different mechanism, but

with comparable efficacy, cations with a high affinity to phosphate, such as Ca 2 + or Mg

2 +, displace basic proteins from the surface.

Negatively charged proteins are believed to bind via their carboxyl groups to the C-sites

at the surface, and proteins with clusters of carboxyl groups are especially strongly

bound. However, such proteins are repelled by the negatively charged apatite surface and

are therefore only weakly retained under standard conditions. They are readily displaced

by phosphate, fluoride or any other anion that binds strongly to calcium. Neutral proteins

are eluted by specific ions such as Mg 2+ F-, phosphate and, to a lesser extent, CI-. The

different retention mechanisms for acidic, neutral and basic proteins facilitate the

development of group separation schemes according to the isoelectric points of the

proteins.

3.6 METHODS FOR THE ANALYSIS OF PROTEIN SIZE, QUANTITATION AND MOLECULAR WEIGHT DETERMINATION:

A universal task involved in protein characterization is purification, quantitation and

identification. Determination of protein size and concentration is a routine procedure in

many research laboratories. For example, protein quantitation is required to calculate and

monitor the protein yield after various enrichment or purification processes as well as to

optimize and standardize downstream experiments such as protein–protein interaction

studies. Protein sizing is commonly used to identify proteins, oligomers and monomers.

27

Some of the techniques used in determination of protein size and concentration are (a)

SDS-PAGE, (b) Lab on a chip assay, (c) Size exclusion chromatography and (d) Mass

spectrophotometry.

3.6.1 SODIUM DODECYLSULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)

Although protein analysis technologies are developing fast, the current standard method

for protein sizing is still denaturing sodium dodecylsulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) [41-43].

Sodium dodecyl sulphate (SDS) is an anionic detergent with a net negative charge. It

binds to most soluble protein molecules in aqueous solutions over a wide pH range. The

amount of bound SDS is proportional to the size of the molecules. The SDS eliminates

most of the complex secondary, tertiary or quaternary structure of proteins, which is one

requirement for protein sizing by SDS-PAGE. Furthermore, it is usually necessary to

reduce protein disulphide bridges before the proteins adopt the random-coil configuration

necessary for separation by size. This is achieved with reducing agents such as 2-

mercaptoethanol or dithiothreitol. In addition, SDS also confers a negative charge to the

polypeptide, which is proportional to its length. This negative charge is utilized to

separate the protein in an electrical field within polyacrylamide gels. The polyacrylamide

forms porous gels allowing to separate molecules by size. A polyacrylamide gel with a

certain acrylamide concentration restrains larger molecules from migrating as fast as

smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS

denatured Polypeptides, the final separation of proteins is dependent almost entirely on

the differences in molecular weight (Mw) of polypeptides. In a gel of uniform pore size

28

the relative migration distance of a protein (Rf) is negatively proportional to the

logarithm of its Mw. If proteins of known Mw are run simultaneously with the

unknowns, the relationship between Rf and Mw can be plotted, and the Mws of unknown

proteins determined.

The sizing accuracy of SDS-PAGE depends on the protein characteristics, such as amino

acid sequence, isoelectric point, structure and the presence of certain side chains or

prosthetic groups. Sizing accuracy may therefore vary for particular proteins, as some

proteins, e.g. glycosylated proteins, are not truly migrating according to their molecular

weight. The sizing precision that can be achieved is approximately 2–7% (see Table 1).

The size resolution can be improved by changing the gel composition to achieve optimal

resolution for the desired experiment.

Protein separations by SDS-PAGE are commonly used to determine the approximate

molecular weights of a protein and the relative abundance of major proteins in a sample.

Usually the relative abundance of proteins within one gel is compared; gel-to-gel compar-

isons are difficult to perform due to the bad reproducibility between gels (see Table 1).

Carefully controlling the experimental conditions for SDS-PAGE such as temperature,

pH, gel composition and staining times allows to significantly improve reproducibility.

However, protein quantitation is therefore preferably performed with colorimetric protein

quantitation assays such as Lowry et al. [44] or Bradford [45]. All commonly used

protein stains used for protein detection exhibits some degree of varying staining

29

efficiency when assaying different proteins, affecting quantitation accuracy (Fig. 1; Table

1).

3.6.2 LAB-ON-A-CHIP TECHNOLOGYThe aim of lab-on-a-chip or microfluidics technology is to shrink processes, in this case

chemical and analytical, to very small dimensions, thus allowing to handle very little

sample volumes. In addition, it has the potential to shorten analysis times significantly

and to automate the process of analysis. The technology allows for the active control of

fluids in microfabricated channels that are only a few micrometers in dimensions without

any moving parts. Typically a number of functional elements are combined on a chip: the

emulation of pumps, valves and dispensers for sample handling on the chip, a separation

column for electrophoretic separation, a reaction system and finally the detection. The

Agilent 2100 bioanalyzer, which was developed in collaboration with Caliper

Technologies (Mountain View, CA, USA), is the first fully commercialized implementa-

tion of microfluidics technology to date [46, 47]. Besides the analysis of RNA and DNA

as well as simple flow cytometric measurements, the multifunctional instrument is also

an alternative tool to characterize various protein samples in respect of size and

quantitation determination.

3.6.3 SIZE EXCLUSION CHROMATOGRAPHY (SEC)Aqueous size exclusion chromatography (SEC), often referred to as gel filtration

chromatography (GFC), is an entropically controlled separation technique in which

molecules are separated based on hydrodynamic volume. SEC is especially well suited

for protein analysis applications such as quantification, impurity testing, reaction

monitoring, product purification, folding studies, and the desalting and exchange of

30

sample buffer. In all but the last of these applications, the protein’s molecular mass may

be simultaneously determined by calibrating SEC column retention times or elution

volumes with an appropriate series of macromolecular standards or by employing

molecular mass sensitive detection methods such as viscosimetry or light scattering. SEC

can handle a very wide range of molecular weights, from several hundred to several

million Dalton, and also an extremely wide range of polarities, from easily water-soluble

to hydrophobic membrane proteins. A 50–100% difference in molecular weight is

typically required. Both, the quantitative data and the molecular weight information are

obtained within a single analysis in good precision and accuracy. SEC does not denature

the separated protein, which can easily be collected for further investigation. The

accuracy of the molecular weight data depends on the calibrant proteins [48], instrument

[49] and software performance [49]. Column calibrations should actually be performed

with macromolecules of the same conformational class as the unknown [48]. When this

prerequisite is fulfilled, the accuracy is typically about 2%. Because proteins of the same

conformational class as the unknown are often not available, water-soluble, globular

proteins with known molecular weight are often used instead. Le Maire et al. [48] list a

series of 15 standard proteins, which were found to be suitable for column calibration.

The molecular weights of uncharacterized proteins can differ significantly from the true

molecular weight due to different elution behavior between the standard and the unknown

proteins. Table 3 summarizes SEC Mw results for proteins of different conformation.

3.6.4 MASS SPECTROMETRYMatrix-assisted laser desorption ionization (MALDI) [50] and electrospray ionization

(ESI) [51] are complementary ionization techniques for mass spectrometry that allow the

31

molecular weight determination of large biomolecules. MALDI in combination with

timeof- flight (TOF) mass analyzers is employed for analyzing peptides/proteins up to

300 kDa for more. A limited dynamic range and a negative influence on sensitivity from

salts or detergents deserve a clean sample for best results. ESI produces multiply charged

ions of biomolecules, therefore even simple and cheap quadrupole mass analyzers with a

mass range up to m/z 3000 allow a correct determination of protein molecular weights.

However, a mathematical algorithm is needed [52,53] to determine the molecular weight

from the mass spectrum, which shows an envelope of multiply charged ions from one

protein (Fig. 4). In the on-line combination with liquid chromatography, proteins up to

about 100 kDa could be injected, chromatographically separated and detected without

tedious sample preparation steps.

In ESI, the number of charge states increases with increasing molecular weight.

Secondly, the more charge states for a protein, the poorer the sensitivity because the

signal is divided over more ions. This explains the limitation in mass to around 100 kDa.

Due to the complexity of overlapping charge envelopes, it may not be possible to

determine the molecular weight for heterogenic proteins without chromatographically

separating the isoforms. Therefore high separation capacity is needed in order to

chromatographically separate protein mixtures/isoforms and avoid overlapping

envelopes. Traditional silica-based reversed phase columns did not allow fast and

efficient protein separations, as mass diffusion in and out the pores was a limiting factor.

The development of Poroshell columns [54] with superficially porous silica-microsphere

packing with a porous outer shell yielded silica-based columns combining the capacity

32

advantage of traditional protein columns and the rapid mass transport of 2-Am non-

porous columns. Typically a water/acetonitrile gradient with up to 0.1% formic acid is

used. The reduced backpressure for superficially porous silica-microsphere packing

allows higher flow rates with constant separation efficiency leading to overall runtimes

as short as 3–5 min (Fig. 5).

In general, this packing material allows a rapid and very efficient separation of large

biomolecules and enables LC/MS with an easy-to-operate and cheap quadrupole

bioanalyzer to be used for protein sizing in a rapid, exact, and sensitive way.

33

3.7 RESTRICTION ENDONUCLEASES:

Restriction endonucleases occur ubiquitously among prokaryotic organism [59, 55].

Their principal biological function is the protection of the host genome against foreign

DNA, in particular bacteriophage DNA [56]. By definition, restriction endonucleases are

part of the restriction modification system which comprises an endonuclease and a

methyltransferase activity. Whereas the substrate of the restriction enzyme is foreign

DNA, which is cleaved in response to defined recognition site, that of the modification

enzyme is the DNA of the host which is modified at the recognition sequence and thereby

protected against attack by the restriction endonuclease [57]. Restriction modification

systems have been classified according to their subunit composition, cofactor

requirement and mode of action and were shown in (Table 4)

Table 2: Classification of Restriction Endonucleases

34

3.7.1 MECHANISM OF ACTION:

The steps involved in recognition of target site by and cleavage of double stranded DNA

are as shown in Figure-2. The reaction cycle starts with the non-specific binding to the

macromolecular DNA, which is followed by a random diffusional walk of the restriction

endonucleases on the DNA. If a recognition site is not too far away from the initial site of

contact it will most likely be located within one binding event. At the recognition site,

conformational changes take place that constitute the recognition process and lead to the

activation of the catalytic centers. This is followed by inline attack of the water molecule

leading to the phosphodiester bond breakage (Figure -3). After phosphodiester bond

cleavage in both stands the product is released either by direct dissociation of the

enzyme-product complex or by a transfer of the enzyme to the non-specific site on the

same DNA molecule [57].

Figure 6: Restriction digestion mechanism: Restriction endonuclease binds to the DNA at a nonspecific site and moved by diffusion walking till in encounters the desired site. Once the site is recognized the confirmation changes takes place with the formation of catalytic site leading to phosphodiester bond breakage [57].

35

Figure 7: Restriction digestion and phosphodiester bond breakage: The phosphodiester bond was cleaved by with the inline attack of water molecule resulting in the formation of 5’ phosphate on one end and 3’ hydroxyl on the other end [58].

3.7.2 DIVALENT CATIONS: Restriction endonucleases as well as many other enzymes that act on phosphate-

containing substrates require Mg2+ or some other similar divalent cation for activity.

Crystallographic studies have found that magnesium ion bind to six ligands: three are

water molecules, two are carboxylates of the enzyme's aspartate residues, and one is an

oxygen atom of the phosphoryl group at the site of cleavage. The magnesium ion holds a

water molecule in a position from which the water molecule can attack the phosphoryl

group and, in conjunction with the aspartate residues, helps polarize the water molecule

toward deprotonation [57].

3.8 AGAROSE GEL ELECTROPHORESIS:Gel electrophoresis is a technique used for the separation of deoxyribonucleic acids,

ribonucleic acids and proteins using an electric current applied to gel matrix [60].

Agarose gel electrophoresis is often used for separation of deoxyribonucleic acids and

ribonucleic acids. Nucleic acids are negatively charged and in the presence of electric

current the DNA moves towards the positive charge. The movement of DNA in the gel

36

will facilitate the separation of DNA based on size. Agarose gel electrophoresis requires

the following.

3.8.1 ELECTROPHRORESIS UNIT: Electrophoresis unit consists of power pack to supply a specific amount of current and to

buffer tank which holds the buffer. Figure 5 is an example for horizontal electrophoresis

unit.

Figure 8: Electrophoresis power pack and gel tank.

3.8.2 AGAROSE: Agarose is a linear polymer composed of alternating residues of D- and L-galactose

joined by -(13) and - (14) glycosidic linkages. The L-galactose residue has an

anhydro bridge between the three and six positions (Figure 6). Chains of agarose form

helical fibers that aggregate into supercoiled structures with a radius of 20-30 nm.

Gelation of agarose results in a three-dimentional mesh of channels whose diameters

range from 50nm to 200 nm.

37

Figure 9: Agarose structure unit

3.8.3 ELECTROPHORESIS BUFFER: Tris-Acetate and EDTA (TAE) and Tris-Boreate (TBE) buffers with (pH 7.8) are the

once commonly used in agarose gel electrophoresis.

3.8.4 LOADING DYE: Gel loading buffers are mixed with the samples before loading into the slot of the gel.

These buffers serve three purposes: They increase the density of the sample, ensuring that

the DNA sinks evenly into the well; they add color to the sample, thereby simplifying the

loading process; and they contain dyes that, in an electric field, move towards the anode

at predictable rates.

3.8.5 ETHIDIUM BROMIDE: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids

and allows the detection of DNA under UV. The difference in the image of DNA

containing gel with and without UV is shown in figure-7.

Figure 10: A. Image of DNA separated gel under visible light and B. image of DNA separated gel under UV.

38

4 MATERIALS AND METHODS

4.1 MATERIALS:

4.1.1 BACTERIAL STRAIN:

4.1.1.1 BACILLUS GLOBIGI:

Bacillus globigi was form ATCC. It expresses BglI and BlgII Methylase and Restriction

endonuclease.

4.1.2 BACTERIAL MEDIA AND AGAR PLATES

4.1.2.1 TRYPTIC SOY BROTH:

4.1.2.2 TRYPTIC SOY AGAR PLATES:

4.1.3 ENZYMES: BglII was form Bangalore Genei (Bangalore,India)

4.1.4 CHEMICALS:Tryptone, Yeast extract, Sodium chloride, Tryptic soy broth, Agar, Acrylamide

Tris Buffer, SDS, Ammonium per sulphate, TEMED, Glacial Acetic Acid, Methanol,

Sodium Thio sulphate, sodium carbonate, silver nitrate, citric acid and protein loading

dye.

4.1.5 PREPARATION OF STOCKS SOLUTIONS:

4.1.5.1 AMPICILLIN (100ΜG/ML):

Dissolve 100mg/ml ampicillin in sterile double distilled water. Storage: store at -20C.

39

4.1.5.2 1.5M TRIS PH 8.8:

Dissolve 18.171gm/100 ml Tris-base in RO-water. Adjusted pH to 8.8 using

concentrated HCl. Store at room temperature.

4.1.5.3 1M TRIS PH 6.8:

Dissolve 12.114gm/100 ml Tris-base in RO-water. Adjusted pH to 6.8 using concentrated

HCl. Store at room temperature.

4.1.5.4 1M TRIS PH 7.5

Dissolve 12.114gm/ 100 ml Tris-base RO-water. Adjust pH to 7.5 using concentrated

HCl. Store at room temperature.

4.1.5.5 1M TRIS PH 8.0

Dissolve 12.114gm/100 ml Tris-base in RO-water. Adjust pH to 8.0 using concentrated

HCl. Store at room temperature

4.1.5.6 0.5M EDTA

Dissolve 18.61 gm of Disodium-EDTA in 80ml of RO-Water. Adjust pH to 8.0 using

concentrated HCl and make up the final volume to 100 ml with RO.

4.1.5.7 5X TRIS GLYCINE BUFFER

Dissolve 1.51gm of Tris Base in 50ml of Ro H2O and add 9.4 gm of Glycine, 5 ml of

10% SDS. Adjust the final volume to 100ml with Ro H2O.

40

4.1.6 BUFFERS:

4.1.6.1 CELL LYSIS BUFFER:

Cell lysis buffer consists of 20 mM Tris pH 6.6/ 7 mM beta mercaptoethanol/ 0.1 mM

EDTA. This was prepared by mixing 1ml of 1M Tris pH 6.6, 27ul of beta

mercaptoethanol and 10ul of 0.5M EDTA and make the final volume to 50ml using Ro-

water.

4.1.6.2 EQUILIBRIATION BUFFER:

Equilibriation buffer consists of 20mM Tris pH 6.6/ 7mM beta mercaptoethanol. This

was prepared by mixing 1ml of 1M Tris pH 6.6 and 27ul of beta mercaptoethanol and

making the final volume to 50ml using Ro-water.

4.1.6.3 WASH BUFFER:

Wash buffer consist of 20mM Tris pH 7.0/ 7mM beta mercaptoethanol. This was

prepared by mixing 1ml of 1M Tris pH 7.0 and 27ul of beta mercaptoethanol and make

the final volume to 50ml using Ro-water.

4.1.6.4 0.3M ELUTION BUFFER:

Dissolve 0.17gm of NaCl in10 ml of wash buffer pH 7.0

4.1.6.5 0.4M ELUTION BUFFER:

Dissolve 0.23gm of NaCl in10 ml of wash buffer pH 7.0

41

4.1.6.6 30% ACRYLAMIDE :

Acrylamide as prepared by dissolve 1 gm of N, N’-Methylene-bis-acrylamide in 30 ml

Ro water by heating at 37C followed by addition of 29 gm of acrylamide. Filter the

solution and store at 4oC.

4.1.6.7 10% SDS:

10% SDS was prepared by dissolve 10gm of Sodium Dodecyl Sulphate (SDS) in 100 ml

of Ro H2O.

4.1.6.8 10% AMMONIUM PER SULPHATE (APS):

10% APS was prepared by dissolve 10gm of Ammonium per sulphate (APS) in 100 ml of

Ro H2O.

4.1.6.9 FIXER:

Fixer was prepared by mix Methanol, Glacial acetic acid and water at a ratio of (4: 1: 5).

4.1.6.10 SODIUM THIO SULPHATE SOLUTION:

Dissolve 0.02 gm of Sodium thiosulphate in 100 ml of Ro H2O.

4.1.6.11 SILVER NITRATE SOLUTION:

Dissolve 0.2 gm Silver Nitrate in 100 ml of Ro H2O. Add 75 µl of Formaldehyde just

before use.

42

4.1.6.12 DEVELOPER:

Dissolve 2 gm of sodium thio sulphate in 100 ml of Ro H2O. Add 50 µl of Formaldehyde

just before use.

4.1.6.13 STOP SOLUTION:

Dissolve 2.1 gm of Citric acid in 100 ml of Ro H2O.

4.1.6.14 1X TRIS GLYCINE BUFFER:

For 100 ml preparation, add 20ml of 5X Tris Glycine Buffer to 80 ml of Ro H2O

4.1.7 EQUIPMENT:Microcentrifgue (eppendorf), Gel documentation system (DNS), Electrophoresis (Tarosn,

India), 37C incubator (Remi, India), Water bath (Remi, India), SDS-PAGE equipment.

4.1.8 PROTEIN MATIX: Sephadex G25 and CM-Cellulose was form Usha biotech, DEAE cellulose was form

Bangalore genei.

43

4.2 METHODS:

4.2.1 GENOMIC DNA ISOLATION: Genomic DNA was isolated using UB-Genomic DNA extraction kit (Usha

Biotech,Hyderabad). Briefly, Glycerol stock of Bacillus globigi was streaked on

Trypticase soy agar plate and incubated at 37c for 18 h. From the plate, single colony

was seeded in 10ml of Trypticase soy broth and cultured at 37C for 18 h. 200 µl of

culture was centrifuged at 10,000 rpm for 30 sec and pellet was washed with 200 µl of

1X PBS. Cells were then resuspended by vortex in 100 µl of Buffer L1. 5ul of lysozyme

(10mg/ml) was added and cells incubated at room temperature for 5min. Cell were again

centrifuged at resuspended in Buffer G1. 5 µl of Buffer G2 and 37 µl of Buffer G3 were

added sequentially with gentle mixing after each addition. 150 µl of Buffer G4 was added

and samples centrifuged at 10,000 rpm for 30 sec. Clear lysate was transferred in to a

fresh eppendorf tube. 20 µl of DBM was added and incubated at room temperature for

1min. DBM was washed once with 200 µl wash buffer-S and DNA was eluted in 50ul of

elution buffer. Elute was transferred into a fresh eppendorf tube and stored at -20C.

4.2.2 ISOLATION OF PLASMID USING MINI PREP KIT: For the isolation of plasmid DNA, glycerol stock of pCI was streaked on LB/Amp plate

and incubated at 37c for 18 h. Single colony from the plates was seeded into 10ml of LB

media containing 100µg/ml Amp. Cultures were incubated in an orbital shaker (REMI) at

37C with shaking. Eighteen hours following incubation plasmid was isolated using UB-

Plasmid Mini Prep Kit. Briefly, 1.5ml of culture was centrifuged at 13,000 rpm for 30 sec

and media was discarded without disturbing the pellet. Bacterial pellet was resuspended

with vortex in Solution 1 (100ul). Solution 2 (200ul) and Solution 3 (150ul) were then

44

added to the cells suspension in sequence with gentle mixing of sample after each

addition. A fluffy precipitate is formed. DNA binding buffer (450ul) was added to the

lysate and centrifuged at 13,000 rpm for 30 sec. Clear lysate was transferred into a fresh

eppendorf tube. DNA binding matrix (DBM) (10ul) was added to the clear lysate and

incubated at room temperature with mixing. Samples were centrifuged at 13,000 rpm for

30sec and clear solution was discarded without disturbing the pellet. DBM was washed

once with wash buffer (500ul) and centrifuged at 13000 rpm for 30 sec. Wash buffer was

discarded without disturbing the pellet. DBM was resuspended in Elution buffer (50ul)

and incubate at room temperature for 2min. Sample was centrifuged at 13000 rpm for 30

sec and elute was transferred in to a fresh eppendorf tube and stored at -20C.

4.2.3 ISOLATION OF RESTRICTION ENDONUCLEASE

4.2.3.1 CULTURE PREPARATION:

Bacillus globigi glycerol stock was streaked on to Trypticase soya agar plate and

incubated at 37C for 18 h. Single colony was seeded into 10ml Tryptic soy broth and

cultured overnight at 37o C in orbital shaker for 16-18 hours. Twohundred microleters of

overnight culture was seeded into 100ml Tryptic soy broth and incubated at 37C in

orbital shaker for 18 hours.

4.2.3.2 HARVESTING OF CULTURE

Culture was collected in to 50ml sterile centrifuge tubes and centrifuged at 5,000 rpm for

5min at 4oC. Clear supernatant was discarded and pellet weight was measured. Pellet was

then resuspended at 40% w/v in cell lysis buffer with vortex.

45

4.2.3.3 ULTRASONIC HOMOGENIZATION:

A biologic ultrasonic homogenizer (Model 300 VT) was used to homogenize Bacillus

globigi cell suspension. A 3.9 mm Titanium micro tip probe was used to homogenize

300-1000ul cell suspension. The probe was immersed into the culture and

homogenization was carried out at Power (40%) in ice for 15 min with 1min incubation

on ice after every 5min pulse. Homogenized cell suspension was centrifuged at 12,000

rpm for 15 min at 4oC and clear lysate was separated for gel filration.

4.2.3.4 GEL FILTRATION:

The clear lysate was passed through a 2ml bed of sephadex G25 which was equilibriated

with 8ml of equilibration buffer before use. 1ml fraction of total protein were collected

after passing the lysate. Once the lysate had entered the bed equilibriation buffer was

added and simultaneously 3 (1ml fractions) were collected.

4.2.3.5 ION EXCHANGE CHROMOTOGRAPHY:

Majority of proteins will be eluted in the first two fractions during gel filtration. First two

fractions were pooled together for ion exchange chromatography (batch mode). CM or

DEAE cellulose equilibrated with the equilibration buffer was added to the gel filtered

sample and protein was allowed to bind to the matrix at room temperature for 30min.

Samples were centrifuged at 12,000 rpm for 2 min at 4oC supernatant was collected into

a fresh tube to analyze the unbound protein. Protein bound CM or DEAE-cellulose was

washed with 2ml wash buffer. Wash was collected into a fresh tube to analyze the loss on

washing. Elution was carried out with high concentration and at different gradients.

46

Eluted samples and intermittent samples were tested for BglII activity by restriction

digestion. Samples with BglII activity were then analyzed by SDS-PAGE.

4.2.4 RESTRICTION DIGESTION:The method is according to sambrook and russle [61]. Restriction digestion was setup

according to the manufacturer’s specifications. The general restriction digestion set up

was as shown below in table.

Table 3: Restriction digestion setup

Component Quantities required for 20 µl reactionControl plasmid Control

EnzymeSamples collected at different points during purification.

DNA (It can vary depending upon the requirement)

3 µl 3 µl 3 µl

10 X Restriction Endonuclease buffer

2 µl 2 µl 2 µl

Enzyme/Fraction ----- 1ul (1u/ug of DNA)

3 µl

BSA (10X) 2 µl 2 µl 2 µlH2O (This varies depending upon the volumes of DNA used )

13 µl 12 µl 10 µl

47

4.2.5 SDS-PAGE:

4.2.5.1 SAMPLE PREPARATION:

Samples were prepared as shown in the Table-4 and heated to 60C for 1 hour in water

bath.

Table 4: Preparation of Gel Loading Samples.

Protein Sample Protease Free Water

Dye

5 µl 5 µl 5 µl5 µl 5 µl 5 µl

4.2.5.2 GEL CASTING:

4.2.5.2.1 PREPARATION OF RESOLVING GEL:Resolving gel was prepared by mix the components given in Table 5.

Table 5: Resolving gel mix

Components for Resolving Gel

Volumes for 12% gel preparation(10ml)

30% Acrylamide 4.0ml1.5mM Tris (pH 8.8) 2.5ml10% SDS 0.1ml10%Ammonium per sulphate 0.1ml TEMED 0.004mlRo H2O 3.3ml

Glass plates were assembled for casting the gel and quickly the gel mix was poured upto

75% volume of the gap between the glass plates. Layer the gel with distilled water and

allow the gel to set for 30 min. Once the gel is set, pour off the water and wash the top of

the gel two times with distilled water to remove any unpolymerized acrylamide.

48

4.2.5.2.2 PREPARATION OF STACKING GEL:Stacking gel was prepared by mixing the components given in Table 6.

Table 6: Stacking gel mix.

Components for stacking gel Volumes for Stacking gel preparation(2ml)

30% Acrylamide 0.33ml1M Tris (pH 6.8) 0.25ml10% SDS 0.02ml10%Ammonium per sulphate 0.02ml TEMED 0.002mlRo H2O 1.4 ml

Once the resolving gel is set stacking gel mix was poured on top of it. A comb was

inserted immediately into the setting stacking gel. Allowed the stacking gel to set for 20

min. Comb can be removed just before running the gel.

4.2.5.3 GEL RUNNING

Mount the gel in the electrophoresis apparatus as specified by the manufacturer and tank

was filled with 1X Tris-Glycine Buffer. Protein marker and the samples were loaded and

gel was run at 200 V for 30 min. Gel was carefully removed from the tank and glass

plates were separated using a spacer. Gel is then placed in a tray and rinsed with distilled

water.

4.2.6 SILVER STAING METHOD:The electrophorosed gel was placed in 5 volumes of fixer and incubated with shaking at

room-temperature for 15 min. Following fixation gel was washed twice with 2 volumes

of water, with 10 min incubation with shaking at room-temperature. Gel was then soaked

in 2 volumes of sodium thio-sulphate solution for 1 min and rinse the gel 2 times with

water. Gel was then placed in 2 volumes of silver nitrate solution for 20min in dark.

49

Following silver staining gel was washed twice with 2 volume of distilled water. Gel was

developed by placing the gel in developer. Once the bands start appearing the reaction

was stopped with the addition of stop solution. Gel was then rinsed and photographed.

4.2.7 AGAROSE GEL ELECTROPHORESIS:The method is according to sambrook and russle [61]. DNA separation was routinely

done in 0.8 to 1% agarose gel in 1 X TAE electrophoresis buffer pH 8.3 (2 mM Tris-

Acetate/0.05M EDTA). Agarose gels were cast in 1 X TAE buffer containing 0.5 g /ml

of ethidium bromide. DNA samples were mixed with 1/6 volume of 6 X loading dye

(Usha Biotech Pvt Ltd, Hyderabad, AP, India) and subjected to electrophoresis under

controlled voltage of 5V/cm. Appropriate DNA size markers (1 kb or 100 bp DNA

ladder) were run alongside the samples to estimate the size of DNA fragments. The DNA

was visualized in an UV transilluminator and gel documentation system (Syngene).

50

5 RESULTS

Restriction endonucleases are one of the most valuable tools in life science research. A

number of these have been identified and characterized form bacteria. They serve as

defense against foreign organisms. BglI and BglII are among the most widely used type

II restriction endonucleases and are expressed in Bacillus globigi. The expression of both

the enzymes in the same host makes the purification of these enzymes complex.

5.1 PURIFICATION OF BglII RESTRICTION ENDONUCLEASE BY ION-EXCHANGE CHROMATOGRAPHY (BATCH METHOD):

Proteins can be selectively isolated by ion-exchange chromatography if their isoelectric

point (pI) is know. However the theoretical pI of majority of proteins might not always be

the true pI of the protein. The pI of BglII is 6.3. In order to overcome the difficulties in

going with the theoretical pI. In here I have tired both anion exchange (DEAE-Cellulose)

and cation exchanger (CM-Cellulose) for the selective binding of BglII.

Bacillus globigi cultures were prepared according to method (4.2.3.1). Culture was

harvested (4.2.3.2) and chromatographic samples were prepared according to methods

(4.2.3.3 and 4.2.3.4).

5.1.1 DEAE-CELLULOSEAnion exchange chromatography was performed using DEAE-cellulose under two pH

conditions (pH 5.6 and pH 8) according to method (4.2.3.5) and samples were analyzed

using restriction digestion (4.2.4) and SDS-PAGE (4.2.5). Restriction digestion pattern of

samples collected at different points of purification were shown in Figure-11. BglII

51

activity was noticed in fraction, supernatant and elutes of pH 8. However none of the

samples in pH 5.6 showed BglII activity. The lack of activity in pH 5.6 samples could be

because of the inactivation of the enzymes at low pH values. The presence of unbound

BglII in supernatant in pH 8.0 after DEAE cellulose addition could be because of the

presence of insufficient DEAE-cellulose.

Figure 11: Purification of BglII using Anionic Exchange Chromatographic Method: Purification was carried out with DEAE cellulose under pH 5.6 (20mM Piperazine Buffer) (lane 1-6) and pH 8.0 (20mM Tris buffer) (lane 7-12). Lane 1 and 7 (supercoiled plasmid), Lane 2 and 8 (Fraction eluted from Sephadex), Lane 3 and 9 (Supernatant collected after DEAE binding), Lane 4 and 10 (20 mM Tris pH 7.5 elution wash), Lane 5 and 11 (Elution with 0.5M Nacl/ 20 mM Tris pH 7.5 elution), Lane 6 and 12 (1:10 Diluted 0.5M elution), Lane 13 (1 Kb ladder)

5.1.2 CM-CELLULOSECation exchange chromatography was performed using CM-cellulose under two pH

conditions (pH 7.2 and pH 2.8) according to method (4.2.3.5) and samples were analyzed

52

1 2 3 4 5 6 7 8 9 10 11 12 13

using restriction digestion (4.2.4) and SDS-PAGE (4.2.5). Restriction digestion pattern of

samples collected at different points of purification were shown in Figure-12. BglII

activity was found in fraction, unbound, wash and elutes at pH 7.2 (Fig 12, lane 2-5).

However there was no activity of BglII at pH 2.8. The lack of activity at pH 2.8 could be

that BglII gets inactivated at low pH.

Figure 12: Purification of BglII using Cationic Exchange Chromatographic Method: Purification was carried out with CM-cellulose under pH 7.2 (20mM Tris buffer) (lane 1-6) and pH 2.8 (20mM Citric Acid Buffer) (lane 7-12). Lane 1 and 7 (supercoiled plasmid), Lane 2 and 8 (Fraction eluted from Sephadex), Lane 3 and 9 (Supernatant collected after DEAE binding), Lane 4 and 10 (20 mM Tris pH 7.5 elution wash), Lane 5 and 11 (Elution with 0.5M Nacl/ 20 mM Tris pH 7.5 elution), Lane 6 and 12 (1:10 Diluted 0.5M elution), Lane 13 (1 Kb ladder)

SDS Page on samples which showed activity with both DEAE-Cellulose and CM-

Cellulose based ion exchange chromatography were shown in Figure 13. DEAE-

Cellulose elutions showed significant enrichment of 15 to 30 kD proteins. BglII falls

under this range and there is a possibility of BglII enrichment using DEAE-Cellulose (pH

53

1 2 3 4 5 6 7 8 9 10 11 12 13

8.0). However, CM-Cellulose chromatography didn’t show any enrichment in proteins.

The low activity of BglII in CM-cellulose sample could be because of the lack of

enrichment.

Figure 13: 12% SDS-PAGE on protein sample collected at different points of protein purification using DEAE-cellulose Chromatography (Tris buffer-pH 7.5) (Lane 1-4) and CM-cellulose Chromatography (Tris buffer-pH 7.2) (Lane 6-9): Lane 1 (Sephadex eluted fraction), Lane 2 (Supernatant collected after DEAE binding), Lane 3 (Wash), Lane 4 (Elution), Lane 5 (Pre stained protein marker), Lane 6 (Sephadex eluted fraction), Lane 7 (7) Supernatant collected after spin, (8) Wash, (9) Elution

54

1 2 3 4 5 6 7 8

5.1.3 OPTIMIZATION OF SALT GRADIENT FOR THE ELUTION OF BglII:

BglII expression was further purified by optimizing the slat gradient using DEAE-

Cellulose chromatographic method (4.2.3.5). Upon binding of BglII to DEAE-cellulose

matrix was divided equally in to 7 tubes and BglII was eluted with 0.1, 0.2, 0.3, 0.4, 0.5M

NaCl. BglII activity in all the samples was tested using restriction digestion (4.2.4) and

the data wash shown in figure 14. BglII was found to be eluted at 0.4 and 0.5M Nacl.

This further reduces the number of host proteins that are eluted along with BglII. The

lack of BglI in these elutes was also confirmed by overnight digestion of all these

samples (figure 15). The lack of BglI activity could be that the pI of BglI is very different

from that of BglII and is not betting bound or eluted under the experimental conditions.

Figure 14: Evaluation of BglII activity in samples collected at different points during DEAE-cellulose chromatography by restriction digestion for 1hour: Lane 1 (1kb ladder), Lane 2 (supercoiled plasmid), Lane 3 (Control for BglII digestion), Lane 4 (crude), Lane 5 (Supernatant collected after DEAE binding), Lane 6 (Fraction-Gel filtered), Lane 7 (1st Wash), Lane 8 (0.1M Nacl/ 20 mM Tris pH 7.5 elution), Lane 9 (0.2 M Nacl/ 20 mM Tris pH 7.5 elution), Lane 10 (0.3M Nacl/ 20 mM Tris pH 7.5 elution), Lane 11 (0.4M Nacl/20 mM Tris pH 7.5 elution) and Lane 12 (0.5M Nacl/ 20 mM Tris pH 7.5 elution).

55

1 2 3 4 5 6 7 8 9 10 11 12

Figure 15: Evaluation of BglII activity in samples collected at different points during DEAE-cellulose chromatography by restriction digestion for 18 hour: Lane 1 (1kb ladder), Lane 2 (supercoiled plasmid), Lane 3 (Control for BglII digestion), Lane 4 (crude), Lane 5 (Fraction-Gel filtered), Lane 6 (Supernatant collected after DEAE binding), Lane 7 (1st Wash), Lane 8 (0.1M Nacl/ 20 mM Tris pH 7.5 elution), Lane 9 (0.2 M Nacl/ 20 mM Tris pH 7.5 elution), Lane 10 (0.3M Nacl/ 20 mM Tris pH 7.5 elution), Lane 11 (0.4M Nacl/20 mM Tris pH 7.5 elution), Lane 12 (0.5M Nacl/ 20 mM Tris pH 7.5 elution)

56

1 2 3 4 5 6 7 8 9 10 11 12

5.2 ISOLATION OF BglII USING THE OPTIMIZED DEAE-CELLULOSE CHROMATOGRAPHY:

Bacillus globigi cultures were prepared according to method (4.2.3.1). Culture was

harvested (4.2.3.2) and chromatographic samples were prepared according to methods

(4.2.3.3 and 4.2.3.4). DEAE-Cellulose chromatography was performed according to the

method (4.2.3.5). The protein bound DEAE was first washed with 0.2M Nacl/20mM Tris

pH 7.5) to get rid of most of the host cell proteins. Proteins bound DEAE-cellulose was

then washed twice with 20mM Tris pH 7.5. BglII was eluted with 0.4M Nacl/20mM Tris

pH 7.5. All the samples collected during this process were tested for BlgII activity using

restriction digestion (4.2.4) and the data was shown in Figure 16. BglII activity was only

seen in crude, fraction and elute. However, BglI activity was also seen in crude and

fraction. The lack of BglI activity in elute indicates that the optimized method was

selective for the isolation of BglII.

57

Figure 16: Purification of BglII from 100ml culture using DEAE-chromatographic method: Lane 1 (1kb ladder), Lane 2 (control plasmid), Lane 3 (Control for BlgII digestion), Lane 4 (crude), Lane 5 (Fraction-Gel filtration), Lane 6 (Supernatant collected after DEAE binding, Lane 7 (1st Wash), Lane 8 (0.2M Nacl/ 20 mM Tris pH 7.5 wash), Lane 9 (2nd Wash), Lane 10 (0.4M Nacl/20 mM Tris pH 7.5 elution).

58

1 2 3 4 5 6 7 8 9 10

6 DISCUSSION

In this project I have optimized the selective isolation of BglII from Bacillus globigi.

BglII is a type II restriction endonucleae and is of importance in life science research.

Compaies like Bioloabs, fermentas, genei and chromus produce these both form their

native source as well as recombinant source. Isolation of BglII form the native source is

difficulty because of the presence of second enzyme BglI which could be purified along

with BglII. Some studies made use of mutants which lack any one of these enzymes there

by facilitating the purification of BglI and BglI [62].

In the current project I have used anionic exchange (DEAE-Cellulose) and cationic

exchange (CM-Cellulose) and slat gradient elution to selectively isolate BglII.

Preliminary studies using DEAE and CM-cellulose showed that DEAE-cellulose is better

for binding BglII (Figure 11 and 12). However, in that study DEAE couldn’t bind 100%

of BglII. 100% of binding in the subsequent experiments was achieved with the use of

more matrix. SDS-PAGE also showed the enrichment of 15-25 kD proteins using DEAE-

Cellulose (Figure 13). BglII being 25kD this result and restriction digestion indicates that

BglII might also have got enriched during the process of DEAE-cellulose

chromatography. pH dose seem to have effect on the activity of BglII. Lower pH (5.6

with DEAE and 2.8 with CM-cellulose) had affected BglII. Digestion was not seen in any

of the samples (crude, fraction, wash and elute) (figure 11 and 12).

Elution of BglII with Nacl gradient (0.1-0.5 M) showed that BglII is selectively eluted at

0.4 M Nacl. This selective elution at 0.4 makes is possible to eliminate host cell proteins

by washing the matrix with 0.2M Nacl there by further purifying BglII. The optimized

59

protocol was fond to be quiet selective for BglII purification. This was represented by the

lack of BglI activity in elutes. Presence of low concentrations of BglI was also ruled out

extending the restriction digestion for longer periods (figure 14 and 15).

60

7 CONCLUSION

The method of selective isolation of BglII restriction endonuclease was optimized. Anion

exchange chromatography using DEAE cellulose at pH 8.0 with salt gradient elution had

resulted in the selective isolation of BglII. The isolated BglII was found to be active with

respect to its restriction endonuclease activity.

61

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