principles of recombinant protein production, extraction ... · bacteria is limited by the...

El-Gayar 2015 Nov 2015, 2(2):18-33

International Journal of Microbiology and Allied Sciences, Nov 2015, Volume 2 Issue 2

International Journal of Microbiology and Allied Sciences (IJOMAS)

ISSN: 2382-5537 November 2015, 2(2):18-33 © IJOMAS, 2015

Principles of recombinant protein production, extraction and

purification from bacterial strains

Khaled E. El-Gayar 1, 2* 1 Department of Biology, Faculty of Science, Jazan University, Kingdom of Saudi Arabia.

2 The Holding Company for Biological Products & Vaccines (VACSERA), Egypt.

*Corresponding Author:

Khaled E. El-Gayar

Department of Biology, Faculty of Science, Jazan University, Kingdom of Saudi Arabia

E-mail: [email protected]

Abstract

Key words: Cloning, E. coli, recombinant protein, production, extraction, purification.

IntroductionRecombinant protein production

The biotechnology field intends to produce

recombinant proteins from bacteria, which

can be made in far greater abundance than

many native proteins [1]. There are a number

of ways through which genetic engineering is

accomplished to produce a recombinant

protein [2]. This process has five main step

included; isolation of the genes of interest,

insertion of the genes into a transfer vector,

transformation of the cells of the organism,

selection of the genetically modified

organism (GMO) from those that have not

been successfully modified [3, 4]. The 5th

Review Article Page: 18-33

Efficient strategies for recombinant proteins production are gaining increasing importance as

more applications that require high amounts of high-quality proteins reach the market. For

example, bacterial hosts are commonly used for the production of recombinant proteins,

accounting for 30% of current biopharmaceuticals on the market. Using biotechnological

methods, it is possible to clone a gene coding for a protein such as insulin and introduce the

cloned fragment into a suitable microorganism using transformation technique, such as E.

coli and Saccharomyces cerevisiae. Escherichia coli expression system continues to

dominate the bacterial expression systems and remain to be the best system for laboratory

research and biotechnological industries. The recombinant microorganism then works as a

living machine to produce a large amounts of proteins. For several reasons, bacteria were the

first microorganisms to be chosen for use as living factories due to their genetics, physiology

and biochemistry. Furthermore, it is easy to culture bacteria in large amounts in inexpensive

and simple media. The recombinant bacteria can grow, multiply very rapidly and produce

heterologous proteins. Finally, we need to extract and purify the resulted heterologous protein

in relatively large quantity for subsequent uses as enzymes, hormones, vaccines, diagnostics

tools, single cell proteins and new proteins for bioremediation.

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important step is gene expression. Where,

gene expression is the process by which the

genetic code the nucleotide sequence of a

gene is used to synthesize the protein. Genes

that code for amino acid sequences are known

as structural genes [5]. Gene expression is

controlled by the joint effect of (i) the global

physiological state of the cell, in particular the

activity of the gene expression machinery,

and (ii) DNA-binding transcription factors

and other specific regulators [6].

Bacteria are particularly convenient for

producing recombinant proteins for

purification purposes. Suitable extraction

methods for bacterial cells include physical

and non - physical methods [7,8]. These

procedures are applicable for preparing

extracts from a variety of Gram-negative

bacteria such as Escherichia coli and

Klebsiella pneumonia [9,10], and Gram-

positive bacteria such as Bacillus subtilis [11].

The production of recombinant proteins in

bacteria is limited by the formation of

cytoplasmic aggregates or inclusion bodies

[12,13]. Pure inclusion bodies were

solubilized using 2 M urea solution at alkaline

pH. The solubilized proteins were refolded

using a re-naturation process and

subsequently purified using chromatographic

procedures [14]. Using bioreactors; it is easy

to culture bacteria in large amounts in

inexpensive and simple media to produce

high-quality proteins reach the market on

large scale under controlled conditions for any

purposes [15-17]. This closed glass vessel has

the adequate arrangement for aeration, mixing

of media by agitation, temperature, pH, anti-

foaming, control of overflow, sterilization of

media and vessel, cooling, and sampling. This

equipment is convenient for operation

continuously for a number of days [18].

Recombinant protein clarification and

extraction

The principals of protein purification is very

simple to remove all contaminants while

retaining as much as possible of the protein of

interest. Contaminants in the extracts of

protein may include a variety of

macromolecules as lipid micelles, nucleic

acids, polysaccharides, and other many

proteins as well as different small molecules.

Small molecules are very easy to separate

from proteins using size selection such as

dialysis, ultrafiltration, and gel filtration.

When macromolecules present in large

amounts; they are more difficult to remove

[19]. So the preparation of an extract

containing the protein in a soluble form

depends on, is this protein secreted

extracellular or intracellular?

Extracellular recombinant proteins

extraction

Extracellular proteins extraction is the

simplest case where the target proteins as

most enzymes are secreted into the culture

media and carried out using:

Centrifugation: Centrifugation is a method

used to separate materials suspended in a

liquid medium depending on the gravity on

particles in suspension [20]. In this method,

denser components of the mixture which

containing soluble proteins migrate away

from the axis of the centrifuge, but less dense

components migrate towards the axis of the

centrifuge [21].

Membrane filtration: Membrane filtration is

used to isolate both cells or debris from

fermentation broth. A membrane is a thin

layer of semi-permeable material that

separates substances when a driving force is

applied through the membrane. The materials

of the membrane may be porous

thermoplastics as Nylon, inorganic oxides as

aluminum oxide and for ultrafiltration,

polysulphones or polyacrylamide [22, 23] .

Ultrafiltration process is used in purification,

desalting and concentration of

macromolecular proteins solutions [19]. Both

of ultrafiltration and microfiltration

separations are based on size exclusion or

particles capture [24].

Intracellular recombinant proteins

extraction The disruption of bacterial cells to extract an

intracellular recombinant protein releases

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protein in soluble form. In the case of

inclusion bodies, accumulations of insoluble

protein that often form in bacteria which have

been induced to express a single protein at a

high level in the bacterial cell. Both chemical

(as enzyme alkali, or detergent treatment) and

mechanical (as pressure cell, sonication, or

homogenizer) are a wide range of disruption

techniques which used in the microbiology

labs [25,26]. A combination of these methods

can lead to a higher protein yield [27].

Disruption by mechanical methods

Homogenization: Homogenization is the

method of converting two immiscible liquids

into an emulsion and considers one of the

common fast methods of disrupting bacterial

cells and decrease the risk to proteins apart

from the release of proteases from any cellular

compartments [28, 29]. This method is

accomplished either by chopping the cells in

a blender, grind, shear, beat and shock or by

forcing the tissue across a narrow opening

between a Teflon pestle and a container [30].

Ultrasonication: Ultrasonication is applying

of sound energy to agitate particles in a

sample, for various purposes. When

frequencies of 20 KHz and above are applied

to solutions, they cause “gaseous cavitation”.

The protein release almost proportional to the

acoustic power input and independent of cell

concentration. The cell paste should be kept

on ice and sonication should be carried out in

bursts of 30 sec or less [28, 31].

Bead mill: Bead mill is an easy method to

shake the suspension of cells, as well as

spores with small glass beads or in a blender

[32, 33]. In general, mechanical methods are

non-specific with higher efficiency and

application broader [34].

Non-mechanical methods of cell disruption

Heat treatment: Heat treatment was used by

heating the microorganisms in an aqueous

acidic solution and then extracting the

proteins with a suitable extracting agent.

Acids preferably used for the pre-treatment

are mineral acids, acetic acid, oxalic acid,

citric acid and formic acid. Suitable extracting

agents include water, aqueous solutions of

inorganic salts, aqueous alkali solutions, for

example, sodium hydroxide and aqueous urea

solutions. Preferred conditions include

heating at from room temperature (25°C) to

about100°C [35, 36].

Freeze-thaw: The freeze-thaw method is

used to lyse bacterial cells. The method

involves freezing a cell suspension in a dry ice

or inside freezer and then thawing the cells at

37°C. This technique of lysis causes cells to

swell and finally break [37, 38].

Osmotic shock: Gram-negative bacteria

requires specific isolation techniques due to

its cell envelope proteins form. It is found that

conventional extraction methods as osmotic

shock cause extracts to be heavily

contaminated with soluble cytoplasmic

proteins [39, 40]. Osmotic shock procedures

are as follow; harvesting the cells from

growth media, suspension the cells in a chilled

neutral buffered solution of high osmotic

pressure (usually containing 20% sucrose),

stand for 30 minutes then centrifugation to

collect the cells and re-suspension the pellet

of the cells in buffer at 4ºC [41].

Lytic enzymes: A number of methods based

on enzymatic means are available for

breaking the cell wall to extract the

recombinant protein product [42]. Enzymatic

methods provide a convenient alternative for

overcoming technical disadvantages of

mechanical disruption [43]. Enzymatic

hydrolysis includes lysozyme hydrolysis,

which cleaves the glucosidic bonds in the

bacterial cell-wall polysaccharide. After that

the inner cytoplasmic membrane can then be

disrupted easily by detergents, osmotic

pressure or any mechanical methods [42]. The

permeability of the cell wall of Gram negative

bacteria can be done using lysozyme with Tris

buffer. This effect can be enhanced by

addition of 1 mM EDTA to chelate the

magnesium ions that stabilize membranes

[44]. Falconer et al., 1997 found that, the

treatment with a combination of the chelating

agent as ≥0.3 mM EDTA and the chaotropic

agent 6 M urea is highly effective at releasing

protein from uninduced E. coli. Also DNase

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and RNase may also be used to enzymatically

de-polymerize DNA and RNA, respectively

[19, 45].

Detergents and solvents: Detergents are

used to release membrane-bound and

intracellular components of any bacterial

cells. Detergents dissociate proteins and

lipoproteins from the cell membrane,

followed by ultracentrifugation [46]. Sodium

lauryl sulphate and triton are common

detergents used [47, 48]. Their action is

depended on pH and temperature. Foaming,

protein denaturation are disadvantages [49].

Preparation of cleared recombinant

bacterial lysates under native conditions

Bacterial culture is centrifuged at 10,000 xg to

get cell pellet. The cells pellet is kept on ice

and re-suspended in lysis buffer (50 mM

NaH2PO4,300 mM NaCl and 10 mM

imidazole) at 2–5 ml per gram wet weight.

One mg/ml lysozyme is added and incubated

on ice for 30 m . After that; sonication on ice

using six 10 s bursts at 200–300 W with a 10

sec cooling period between each burst is done.

Because of the lysate is very viscous, so 10

µg/ml RNase and 5 µg/ml DNase are added

then incubated on ice for 10–15 min. The

lysate is Centrifuged at 10,000 xg for 20–30

min at 4°C to precipitate the cellular debris

[25, 50].

Protein precipitation

It is possible to partially purify a protein from

a mixture by adding a precipitating agent. It is

used as a separation step through the early

stages of a purification procedure followed by

chromatographic separations steps. Also

precipitation can be used as a method for

protein concentration prior to purification

steps.

Precipitation by alteration of the pH: The

protein solubility depends on the pH of the

solution. Any protein can be either positively

or negatively charged due to the terminal

amine -NH2 and carboxyl -COOH groups

[51]. It is positively charged at low pH and

negatively charged at high pH. The

intermediate pH at which a protein molecule

has a net charge of zero is called the

isoelectric point of that protein. Adjusting the

pH of the solution to close or equal to the

isoelectric precipitation (pI) of the protein

considers one of the easiest methods of

precipitating a protein and achieving a degree

of purification. pI is often used to precipitate

unwanted proteins, rather than to the protein

of interest [52].

Precipitation by altering the ionic strength:

Lowering the ionic strength can precipitate

some proteins. Ionic strength reducing agents

are organic compounds that decrease the ionic

strength of aqueous salt solutions. Ionic

strength reducing agents may have strong

effects in chromatographic methods,

precipitation and thus crystallization itself

[53]. The precipitated protein is usually not

denaturated and activity is recovered upon re-

dissolving the pellet. In practice ammonium

sulphate that salt solutions with high ionic

strength is the most commonly used salt.

Ammonium sulphate is cheap, and

sufficiently soluble; a saturated ammonium

sulphate solution in pure water is

approximately 4M [54, 55]. When high

concentrations from highly charged ions such

as ammonium sulfate are added to bacterial

lysate, these groups compete with the proteins

to link the water molecules. This removes the

water molecules from the protein and causes

decreasing in its solubility to precipitate

proteins. There are some factors affect the

concentration at which a particular protein

will precipitate include; the number and

position of polar groups, protein molecular

weight, pH of the solution and temperature at

which the precipitation is done [56].

Precipitation of proteins from solutions are

carried out by dissolving ammonium sulfate

into the protein solution with stirring at 0°C to

avoid proteins denaturation. Table (1) shows

the weight per grams of ammonium sulfate to

be added to one liter of solution to produce a

change in the concentration (% saturation) of

ammonium sulfate [57, 58].

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Table 1: Use of ammonium sulfate concentration table. 1- Selection of the initial

concentration of ammonium sulfate (%) from saturation from the left column. 2-

Selection of required final concentration of ammonium sulfate (%) from saturation from

the top row. Intersection will give an exact amount of ammonium sulfate in grams for 1

liter of initial solution. Adopted from [57].

% 10 15 20 25 30 33 35 40 45 50 55 60 65 70 75 80 85 90 95 100

0 56 84 114 144 176 196 209 243 277 313 351 390 430 472 516 561 610 662 713 767

10 28 57 86 118 137 190 183 216 251 288 326 365 406 449 494 540 592 640 694

15 28 57 88 107 120 153 185 220 256 294 333 373 415 459 506 556 605 657

20 29 59 78 91 123 155 189 225 262 300 340 382 424 471 520 569 619

25 30 49 61 93 125 158 193 230 267 307 348 390 436 485 533 583

30 19 30 62 94 127 162 198 235 273 314 356 401 449 496 546

33 12 43 74 107 142 177 214 252 292 333 378 426 472 522

35 31 63 94 129 164 200 238 278 319 364 411 457 506

40 31 63 97 132 168 205 245 285 328 375 420 469

45 32 65 99 134 171 210 250 293 339 383 431

50 33 66 101 137 176 214 256 302 345 392

55 33 67 103 141 179 220 264 307 353

60 34 69 105 143 183 227 269 314

65 34 70 107 147 190 232 275

70 35 72 110 153 194 237

75 36 74 115 155 198

80 38 77 117 157

85 39 77 118

90 38 77

95 39

Precipitation with ethanol: The miscible

organic liquids as acetone or ethanol is one of

the most common types of precipitating

agents. Ethanol is more efficient for proteins

with surfaces that are almost dominated by

polar amino acid side chains and other

hydrophilic [59]. To precipitate protein from

solution using ethanol, one volume from

solution is mixed with 9 volumes from cold

absolute ethanol and storage at -20◦C

overnight. To collect proteins, the samples are

centrifuged at 10000 xg for 20 min at 4◦C and

the supernatant is removed. The pellet is

washed once with absolute ethanol before it is

dried [60].

Recombinant Protein purification

It is found that a minor protein may need

many purification procedures and high skills

on the purification methods but a major

protein is not so difficult to be purified.

Chromatography is the most used method in

protein purification. The basic of

chromatographic purification is distribution

of separated protein molecules between two

immiscible phases named mobile and

stationary phase. Chromatographic methods

are classified by physical shape of stationary

phase, nature of mobile and stationary phase

and mechanism of separation. So the

chromatographic methods are named

depending on their popularity [49].

The methods of protein purification

Th

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ntr

ati

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of

am

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Required final concentration of ammonium sulfate )%(

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Dialysis: Dialysis is known as the process of

separating molecules in solution by the

difference in their diffusion rates through a

semipermeable membrane as dialysis tubing

[61]. Generally, in life science research, the

most common application of dialysis is to

remove the undersides small molecules as

salts, reducing agents or dyes from larger

macromolecules as DNA, polysaccharides or

proteins [62]. In protein purification, it is

important to remove salts or change the buffer

from any step in the protein purification to the

next step. This is achieved by dialysis. The

protein solution is placed in a bag semi-

permeable membrane and placed in the

required buffer as phosphate buffer saline,

small molecules can pass across the

membrane freely whilst large molecules are

retained. The semipermeable dialysis tubing

is usually made of cellulose acetate, with

pores of between 1-20 nm in diameter [63].

Dialysis is often carried out overnight against

buffer, usually at 4ºC to minimize losses in

activity [49]. Dialysis tubing is prepared by

cutting into pieces of convenient length (10 to

30 cm), soaked in distilled water for 10

minutes after which the pieces are soaked in

50% ethanol for about 10 minutes. Then the

dialysis tubing is boiled in a solution of 2%

sodium bicarbonate and 1 mM EDTA for 15

minutes. The tubing was allowed to cool then

stored in 50% ethanol at 4◦C. Before use, the

tubing is carefully washed from inside and

outside with distilled water then with the

buffer to be used [64].

Gel filtration chromatography: In gel

filtration, protein molecules in solution are

separated according to the difference in their

sizes as they pass through a column packed

with a chromatographic gel medium [49]. The

various media that can be used for this

purpose are spherical beads composed of

matrices containing pores. When a mixture of

different sized molecules are placed on top of

a column containing these beads, the larger

molecules cannot easily diffuse into the pores

and are eluted 1st from the column with no

resistance. But the small molecules diffuse

into the beads in the gel beads. There are

many types of gel filtration columns as

Sephacryl high resolution (HR), Superdex,

Sephadex, Superose and sepharose [65] . Also

gel filtration is used in desalting and buffer

exchange. A gel filtration matrix with a small

pore size (e.g. Sephadex G-25) is poured into

a column to give a bed volume of

approximately five times the volume of

sample to be desalted [66]. To purify protein

using gel filtration: For example, enzyme is

purified through gel filtration columns using

Sephadex G-75 as gel filtration resin.

Sephadex G-75; 5 g is suspended in excess of

buffer (50 mM Tris-HCl, pH 7.5 containing

10 mM CaCl2) to be swelled. The swelling

process is carried out as follow; The slurry is

poured carefully into (1.5 × 30cm) column

with the aid of a glass rod. The bed height is

adjusted to 30 cm by settling the gel beads.

The column is then washed and equilibrated

with buffer at a flow rate of 36 ml / hour using

a peristaltic pump. The dialyzed protein is

applied to the Sephadex G-75 column with the

aid of an adaptor. The enzyme is eluted with

50 m M Tris HCl buffer, pH7.5 containing 10

mM CaCl2 at a flow rate of 36 ml/hour.

Fractions (3 ml) are collected after which the

absorbance at 280 nm and the enzyme activity

are assayed. Active fractions are collected and

re-precipitated on ice with solid ammonium

sulfate [67-69]

Affinity chromatography: Affinity

chromatography is an adsorption

chromatography method of separating

biochemical mixtures based on a highly

specific interaction where the molecule to be

purified is specifically and reversibly

adsorbed by binding substance (ligand)

immobilized on an insoluble support (matrix)

as that between antigen and antibody, enzyme

and substrate, hormones and receptors, lectin

and polysaccharides or nucleic acids and

histones [70]. Affinity purification is often of

the order of several thousand fold and

recoveries of active material are generally

very high. For this reason, affinity

chromatography can be used for purifying

substances from complex biological mixtures,

separating native from denatured forms of the

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same substances and removing small amounts

of biological material from large amounts of

contaminating [49]. The preparation of a

number of agarose and polyacrylamide bead

derivatives useful in the purification of

proteins by affinity chromatography is

described. These techniques permit (a) The

attachment of ligands to the gel through

extended hydrocarbon chains which place the

ligand at varying distances from the gel

matrix backbone; (b) The covalent attachment

of ligands to agarose or polyacrylamide gels

through amino, carboxyl, phenolic, or

imidazole groups of the ligand; and (c) The

preparation of adsorbents containing ligands

attached by bonds which are susceptible to

specific chemical cleavage, thus providing

means of removing the intact protein-ligand

complex from the affinity adsorbent.It is

demonstrated that successful application of

affinity chromatography in many cases will

critically depend on placing the ligand at a

considerable distance from the matrix

backbone [71]. Ni-NTA Agarose is an affinity

chromatography matrix to purify recombinant

proteins carrying a His tag. Histidine residues

in the His tag bind to the vacant positions in

the coordination sphere of the immobilized

nickel ions with high specificity and affinity.

To purify recombinant antigens from bacterial

lysate after dialysis: One ml of the 50% Ni-

NTA slurry is added to 4 ml cleared lysate and

mixed gently by shaking at 200 rpm on a

rotary shaker at 4°C for 60 min. The lysate Ni-

NTA mixture is loaded into a column with the

bottom outlet capped. Bottom caps were

removed and the column flow-through was

collected. Five µl from flow through were

saved for SDS-PAGE analysis. Wash twice

with 4 ml wash buffer (50 mM NaH2PO4,

300mM NaCl, 20 mM imidazole, the pH

adjusted to 8.0) is done followed with

collection the washing fractions for SDS-

PAGE analysis. Finally, the protein is eluted

4 times with 0.5 ml elution buffer for each

tube (the elution buffer contains;50 mM

NaH2PO4,300mM NaCl, 250 mM imidazole,

the pH adjusted to 8.0) then analyzed by SDS-

PAGE [25].

Ion Exchange Chromatography: Ion

exchange is defined as the reversible

exchange of ions in solution with ions

electrostatically bound to some sort of

insoluble support medium. The ion exchanger

is the inert support medium to which are

covalently bound positive (in case of an anion

exchanger) or negative (in case of a cation

exchanger) functional groups [19]. The pH of

the buffer selected for binding and elution

affects the charge on weak ion exchangers but

not on strong ion exchangers which their

charge over a wide pH range [72]. To prepare

ion exchanger column; DE-52 anion

exchanger for example 15g is suspended in

excess of buffer (50 m M Tris- HCl, pH 7.5)

in order to be swelled. The slurry is poured

carefully into a (2.7×6 cm) column with the

aid of a glass rod. The addition of the gel

suspension is continued until a bed height of

6 cm. The column is then washed and

equilibrated with buffer at a flow rate of 72

ml/hr using a peristaltic pump. Ten ml of the

dialyzed protein is applied to the DE-52

column with the aid of an adaptor. The protein

is eluted with 50 mM Tris-HCl buffer, pH 7.5

then 50 mM Tris-HCl buffer, pH 7.5

containing 0.5 M NaCl respectively at a flow

rate of 72 ml/hour. Fractions (6ml) are

collected after which the absorbance at 280

nm and the enzyme activity are determined.

Active fractions are collected and re-

precipitated with solid (NH4)2 SO4 (65%)

saturation.

So, from above it can be purified both of

extracellular and intracellular protein after

extraction from bacteria using dialysis and

chromatography. For example, Bacillus

subtilis extract (Cell-free supernatant) is

obtained by precipitating cells using

centrifugation at 8,000 rpm. Solid ammonium

sulfate is added to the supernatant to reach

65% saturation as showed in Table 1. The

precipitate is removed by centrifugation at

12,000 rpm for 30 minutes at 4°C. Pellets are

re-suspended in 0.1 M Tris-HCl buffer, pH

7.5containing 10 mM CaCl2, and dialyzed

overnight against the same buffer. Samples

are then taken to determine protein content

and proteolytic activity. The protein is further

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purified using anion exchange DE-52 column

followed by Sephadex G-50 gel filtration

column [73].

Protein content estimation

Bradford method considers the most used

protocol to estimate pure protein [74]. The

assay is based on the observation that the

absorbance maximum for an acidic solution of

Coomassie Brilliant Blue G-250 shifts from

465 nm to 595 nm when binding to protein

occurs. Both ionic and hydrophobic

interactions stabilize the anionic form of the

dye causing a change in the visible color [75].

In this protocol; Bovine serum albumin; BSA

stock (1mg/1ml) is prepared for standard

curve. Also PBS (phosphate buffer saline) is

prepared. 100 mg Coomassie Brilliant Blue

G-250 is Dissolved in 50 ml 95% ethanol, and

100 ml 85% (w/v) phosphoric acid is added

then this mix diluted to 1 liter when the dye

has completely dissolved, and filtered through

Whatman #1 paper just before use. Standards

containing a range of 5 to 100 micrograms

protein (BSA) in 100 µl volume. 5 ml dye

reagent is added to BSA standards and

unknown samples separately and incubated 5

min then the absorbance at 595 nm is

measured. A graph is drawn using the X-axis

for standard protein concentration and Y-axis

for optical density at 595 nm using a

spectrophotometer program which calculate

the protein content of the samples

immediately using the standard curve of the

graph or it can be calculated manually protein

[74].

In another method, soluble proteins is carried

out according to Lowry et al.,1951 [76]. The

method combines the reactions of cupper ions

with the peptide bonds under alkaline

conditions (the Biuret test) with the oxidation

of aromatic protein residues. Protein

concentrations of 0.01–1.0 mg/mL can be

estimated by the Lowry method and is based

on the reaction of cupper; Cu+, produced by

the oxidation of peptide bonds, with Folin –

Ciocalteu reagent (a mixture of

phosphotungstic acid and phosphomolybdic

acid in the Folin – Ciocalteu reaction). The

reaction mechanism involves reduction of the

Folin–Ciocalteu reagent and oxidation of

aromatic residues (mainly tryptophan and

tyrosine). Cysteine is also reactive to the

reagent. Therefore, cysteine residues in

protein also contribute to the absorbance seen

in the Lowry Assay [77]. Four hundred of

appropriately diluted crude protein sample is

added to 0.5 ml protein assay solution (25ml

5 % (w/v) Na2CO3, 0.5 ml 1% (w/v) CuSO4

and 0.5 ml 2 % (w/v) sodium potassium

tartarate). The tubes are mixed well by

inversion and allowed to stand for 10 minutes

at room temperature after which 0.1 ml of 2N

Folin reagent is added. After 30 minutes, the

developed color is measured at 750 nm. A

standard curve is established each time using

BSA.

Protein electrophoresis

Gel electrophoresis is a method used to

separate and analyze macromolecules as

DNA, RNA and proteins and their fragments,

based on their size and electric charge. It is

used in clinical chemistry, biochemistry and

molecular biology to separate proteins by

charge and size [78].

Proteins are amphoteric compounds; their net

charge therefore is determined by the pH of

the medium in which they are suspended. In a

solution with a pH above its isoelectric point,

a protein has a net negative charge and

migrates towards the anode in an electrical

field. Below its isoelectric point, the protein is

positively charged and migrates towards the

cathode [79]. Polyacrylamide gel

electrophoresis (PAGE) is used for separating

proteins ranging in size from 5 to 2,000 kDa

due to the uniform pore size provided by the

polyacrylamide gel. Pore size is controlled by

modulating the concentrations of acrylamide

and bis-acrylamide powder used in creating a

gel [80]. To separate proteins under denature

condition; sodium dodecyl sulfate (SDS) must

be added. SDS is an anionic detergent which

denatures proteins by "wrapping around" the

polypeptide backbone. SDS confers a

negative charge to the polypeptide in

proportion to its length. It is usually necessary

to reduce disulphide bridges in proteins before

they adopt the random-coil configuration

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necessary for separation by size: this is done

with 2- mercaptoethanol or dithiothreitol. In

denaturing SDS-PAGE separations therefore,

migration is determined not by intrinsic

electrical charge of the polypeptide, but by

molecular weight [81].

Sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) is performed as

described earlier [82]. The glass plates and

spacers are assembled onto casting stand. The

running gel, (10%) is prepared by mixing 3.4

ml of (30%) stock acrylamide/bis solution

(acrylamide, 29.2g; bisacrylamide, 0.8g; in

100ml distilled water), 2.0 ml of 1.5 M Tris -

HCl, pH 8.8, 4.45 ml of distilled water and 0.1

ml of 10% SDS. Tetra-methylene diamine

(TEMED) and ammonium persulfate (APS)

are added just before use at a final

concentration of 0.5% (v/v) and 1% (w/v)

respectively. The above mixture are poured

using Pasteur pipette in an assembly unit (10×

10 cm) and overlaid carefully with

isopropanol. The gel is allowed to be

polymerized at room temperature for about 15

minutes. After polymerization completed, the

overlaying layer is poured off and the top of

the gel is washed with distilled H2O. Stacking

gel (5%) is prepared by mixing 0.68 ml of

(30%) acrylamide / bis stock solution, 0.05 ml

10 % SDS, 0.5 ml 0.6 M Tris-HCl, pH 6.8 and

3.75 ml of distilled water. TEMED and APS

are added at concentrations of 0.5% (v/v) and

1% (w/v) respectively, while polymerization

is carried out as above. The ten teeth comb is

inserted in the stacking gel but with ◦45 angle

to avoid formation of air bubbles under the

teeth of the comb. The gel is poured using a

Pasteur pipette and then the comb is pushed to

fit into its place. After polymerization the

comb is removed from the gel and wells are

washed carefully with reservoir buffer. The

gel is installed to the reservoir buffer solution

containing 0.05 M Tris-HCl, pH 8.3, 0.384 M

glycine, and 0.1 % (w/v) SDS .Samples are

prepared by mixing small volume of protein

sample (100g) with 2X sample application

buffer (SAB) [0.6 M Tris - HCl, pH 6.8, 1%

SDS, 10% - mercaptoethanol, 10% glycerol,

and 0.05% bromophenol blue], then boiled at

95◦C in water bath for 3 minutes. Samples are

applied to the slab gel along with SDS

molecular weight marker (10-250 K Dalton).

Electrophoresis is carried out at a constant

current 15 mA for about 1.5 hours. As the

front line of the run is near the end of gel, the

current is stopped and the gels are removed

from the tank. Gels are stained with

Coomassie blue [0.1% Coomassie Brilliant

Blue R-250, in 50% methanol and 10% glacial

acetic acid] for 2 hours with gentle shaking at

room temperature. The gels are de-stained

using a de-stainer solution (100 ml methanol,

70 ml glacial acetic acid and 830 ml distilled

water) and then incubated with changes of de-

stainer until clear background of the gel

obtained.

Determination of Molecular Weight

This is done by SDS-PAGE of proteins or

agarose gel electrophoresis of nucleic acids of

known molecular weight along with the

protein or nucleic acid to be characterized. For

protein, a linear relationship exists between

the logarithm of the molecular weight of an

SDS-denatured polypeptide and its Rf then

read off the log Mr of the sample after

measuring distance migrated on the same gel.

As shown in Figure (1); the Rf is calculated

as the “ratio of the distance migrated by the

molecule to that migrated by a marker dye-

front” [83, 84].

Figure 1: Calculation the unknown protein

molecular weight by drawing a linear

relationship exists between the logarithm

of the molecular weight of an SDS-

denatured polypeptide and their Rf.

Preparing a Purification Table

A purification summary table should allow a

researcher to evaluate the procedure and

readily detect particularly effective and

26

El-Gayar 2015 Nov 2015, 2(2):18-33


Table 2: Purification table of any enzyme produced by the recombinant bacteria.

Purification step Volume

(ml)

mg protein

/ml

Units/

ml

Total units Sp.

Activity

Fold

Purification

Cell- free supernatant

Pellet after dialysis

After DE-52 column

ineffective purification steps. According to

(Burgess,2009) A suitable table will be

contained the following columns [55]:

The main steps in the purification: These

include steps like; the result of cell disruption

(Crude lysate) after centrifugation,

ammonium sulfate precipitation, pooling peak

from an ion exchange column, gel filtration

column or from an affinity column,

concentration then dialyzing [85].

Amount of total protein (mg): This is

usually determined by any standard protein

assay such as Bradford method as mentioned

above.

Enzyme activity determination: For each

major steps; enzyme assay for the target

protein should be carried out [86].

Specific activity (units/mg): It is calculated

by dividing the total activity (units) by the

total protein (mg) [87].

Overall yield (%): The yield at a step in the

procedure is known as the amount of target

(total target protein or total activity) at that

step divided by the amount of target in the

first step [55].

Purity of target protein (%): In case of

another proteins not enzymes; Purity is

determined by scanning a stained SDS–PAGE

and measuring the amount of the stain

associated with the target band as a fraction of

the stain associated with all the bands on the

gel [88].

Relative or fold purification: This is setting

the initial purity at a value of one and then

giving the purity at each step relative to that

of the first step [89]. Table (2) shows all

purification steps of any enzyme produced

from recombinant Bacteria. The final step

represents an overall fold purification [73].

Storage of proteins

It is essential that, the pure target protein

maintains its original functional behavior over

an extended period of storage which may

reach to years [90]. Under the same set of

external conditions (as pH, temperature and

buffer composition) there are some proteins

those appear very stable at one stage in

purification then lose stability at the next

stages. Thus, stability as enzyme activity

needs to be monitored at every purification

step [19]. There are general precautions to

achieve the stability for any protein.

Immediately after extraction, adding protease

inhibitors. One of the key ingredients in many

protease inhibitor cocktails is PMSF (phenyl

methyl sulfonyl fluoride), a commonly used

protease inhibitor that binds covalently to

active site serine residues on serine proteases

(trypsin, chymotrypsin, thrombin, subtilisin,

etc.), permanently inactivating them [91]. A

metal chelator,1-10 mM EDTA is used to bind

heavy metals that, when free, can “poison”

sensitive enzymes, activate certain

metalloproteases, or enhance sulfhydryl

oxidation. The reducing agents

mercaptoethanol or dithiothreitol (DTT)may

also be added at low concentration (1mM) to

prevent protein oxidation. Sodium azide

(0.02%) prevents growth of microorganisms

[19, 92]. Filtration with a filter of a pore size

0.22 µm will exclude all microbe. The

inclusion of low molecular weight substances

as glycerol or sucrose in protein solution can

greatly stabilize the protein’s biological

activity. Refrigeration at 4-6ºC is often

27

El-Gayar 2015 Nov 2015, 2(2):18-33


sufficient for the preservation of a protein’s

biological activity [44]. Sometimes, however,

it may be necessary to use a low temperature

laboratory freezer designed to maintain

temperatures in the range of –70 to -80ºC [93].

The process of isolating a solid substance

from solution by freezing the solution and

evaporating the ice under vacuum. Many

microorganisms and proteins survive

lyophilization well, and it is a favored method

of drying vaccines, pharmaceuticals, blood

fractions and diagnostics [44].

Conclusion This study provides a system for recombinant

protein extraction and purification from an

expression system as E. coli. Production of

pure recombinant proteins at high yield solved

many problems in the fields of

bioremediation, medicine, agriculture,

nutrition and industry.

Acknowledgment I am thankful to Department of Biology,

Faculty of Science, Jazan University, KSA

and The Holding Company for Biological

Products & Vaccines (VACSERA), Cairo,

Egypt for provision of expertise, and technical

support in the implementation.

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El-Gayar 2015 Nov 2015, 2(2):18-33


For Citation:

El-Gayar KE. 2015. Principles of recombinant protein production, extraction and

purification from bacterial strains. International Journal of Microbiology and Allied

Sciences. 2(2):18-33.

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