Abstrak

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.

Introduction
Recombinant 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 5 th
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 Grampositive 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, antifoaming, 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 pro 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 (25C) to about100C [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 37C. 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 4C [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 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 25 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 g/ml RNase and 5 g/ml DNase are added then
incubated on ice for 1015 min. The lysate is Centrifuged at 10,000 xg for 2030
min at 4C 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 redissolving 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 0C 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].
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 -20C overnight. To collect proteins, the samples are centrifuged at
10000 xg for 20 min at 4C 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
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 semipermeable 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 semi- permeable 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 4C
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 4C. 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 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% NiNTA slurry is added to 4 l cleared lysate
and mixed gently by shaking at 200 rpm on a rotary shaker at 4C for 60 min. The
lysate NiNTA mixture is loaded into a column with the bottom outlet capped. Bottom
caps were removed and the column flow-through was collected. Five l 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 SDSPAGE 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 SDSPAGE [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 (2.76 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 reprecipitated 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 4C. 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 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 l 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 0.011.0 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 hosphomolybdic acid in the Folin Ciocalteu reaction).
The reaction mechanism involves reduction of the FolinCiocalteu 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, ased 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 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 95C 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 destainer 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 dyefront [83, 84].

Preparing a Purification Table

A purification summary table should allow a researcher to evaluate the procedure
and readily detect particularly effective and 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 SDSPAGE 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 m will exclude all
microbe. The inclusion of low molecular weight substances as glycerol or sucrose in
protein solution can greatly stabilize the proteins biological activity. Refrigeration at
4-6C is often sufficient for the preservation of a proteins 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 -80C [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