Principles of recombinant protein production, extraction and ...

El-Gayar 2015

Nov 2015, 2(2):18-33

International Journal of Microbiology and Allied Sciences (IJOMAS) ISSN: 2382-5537 November 2015, 2(2):18-33 © IJOMAS, 2015 Review Article

Page: 18-33

Principles of recombinant protein production, extraction and purification from bacterial strains 1

Khaled E. El-Gayar 1, 2* 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 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. Key words: Cloning, E. coli, recombinant protein, production, extraction, purification.

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 5th

International Journal of Microbiology and Allied Sciences, Nov 2015, Volume 2 Issue 2 18

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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 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 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].

example, sodium hydroxide and aqueous urea solutions. Preferred conditions include heating at from room temperature (25°C) to about100°C [35, 36].

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

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].

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].

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 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 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. 2Selection 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].

The initial concentration of ammonium sulfate )%(

Required final concentration of ammonium sulfate )%( % 10 15 0 56 84 28 10 15 20 25 30 33 35 40 45 50 55 60 65 70 75 80 85 90 95

20 114 57 28

25 144 86 57 29

30 176 118 88 59 30

33 196 137 107 78 49 19

35 209 190 120 91 61 30 12

40 243 183 153 123 93 62 43 31

45 277 216 185 155 125 94 74 63 31

50 313 251 220 189 158 127 107 94 63 32

55 351 288 256 225 193 162 142 129 97 65 33

60 390 326 294 262 230 198 177 164 132 99 66 33

65 430 365 333 300 267 235 214 200 168 134 101 67 34

70 472 406 373 340 307 273 252 238 205 171 137 103 69 34

75 516 449 415 382 348 314 292 278 245 210 176 141 105 70 35

80 561 494 459 424 390 356 333 319 285 250 214 179 143 107 72 36

85 610 540 506 471 436 401 378 364 328 293 256 220 183 147 110 74 38

90 662 592 556 520 485 449 426 411 375 339 302 264 227 190 153 115 77 39

95 713 640 605 569 533 496 472 457 420 383 345 307 269 232 194 155 117 77 38

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].

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].

Recombinant Protein purification

The methods of protein purification


100 767 694 657 619 583 546 522 506 469 431 392 353 314 275 237 198 157 118 77 39

El-Gayar 2015 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 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

Nov 2015, 2(2):18-33 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% NiNTA 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 NiNTA 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 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 (2.7×6 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 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 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 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].

Figure 1: Calculation the unknown protein molecular weight by drawing a linear relationship exists between the logarithm of the molecular weight of an SDSdenatured 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


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

Volume

mg protein

Units/

(ml)

/ml

ml

Total units

Sp.

Fold

Activity

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]:

from recombinant Bacteria. The final step represents an overall fold purification [73].

Storage of proteins The main steps in the purification: These It is essential that, the pure target protein include steps like; the result of cell disruption maintains its original functional behavior over (Crude lysate) after centrifugation, an extended period of storage which may ammonium sulfate precipitation, pooling peak reach to years [90]. Under the same set of from an ion exchange column, gel filtration external conditions (as pH, temperature and column or from an affinity column, buffer composition) there are some proteins concentration then dialyzing [85]. those appear very stable at one stage in Amount of total protein (mg): This is purification then lose stability at the next usually determined by any standard protein stages. Thus, stability as enzyme activity assay such as Bradford method as mentioned needs to be monitored at every purification above. step [19]. There are general precautions to Enzyme activity determination: For each achieve the stability for any protein. major steps; enzyme assay for the target Immediately after extraction, adding protease protein should be carried out [86]. inhibitors. One of the key ingredients in many Specific activity (units/mg): It is calculated protease inhibitor cocktails is PMSF (phenyl by dividing the total activity (units) by the methyl sulfonyl fluoride), a commonly used total protein (mg) [87]. protease inhibitor that binds covalently to Overall yield (%): The yield at a step in the active site serine residues on serine proteases procedure is known as the amount of target (trypsin, chymotrypsin, thrombin, subtilisin, (total target protein or total activity) at that etc.), permanently inactivating them [91]. A step divided by the amount of target in the metal chelator,1-10 mM EDTA is used to bind first step [55]. heavy metals that, when free, can “poison” Purity of target protein (%): In case of sensitive enzymes, activate certain another proteins not enzymes; Purity is metalloproteases, or enhance sulfhydryl determined by scanning a stained SDS–PAGE oxidation. The reducing agents and measuring the amount of the stain mercaptoethanol or dithiothreitol (DTT)may associated with the target band as a fraction of also be added at low concentration (1mM) to the stain associated with all the bands on the prevent protein oxidation. Sodium azide gel [88]. (0.02%) prevents growth of microorganisms Relative or fold purification: This is setting [19, 92]. Filtration with a filter of a pore size the initial purity at a value of one and then 0.22 µm will exclude all microbe. The giving the purity at each step relative to that inclusion of low molecular weight substances of the first step [89]. Table (2) shows all as glycerol or sucrose in protein solution can purification steps of any enzyme produced greatly stabilize the protein’s biological activity. Refrigeration at 4-6ºC is often International Journal of Microbiology and Allied Sciences, Nov 2015, Volume 2 Issue 2 27

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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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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.
