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  • E.coli Cell Lysis
  • Purification Overview
  • Affinity Chromatography
  • Ion Exchange Chromatography
  • Gel Filtration

E.coli Cell Lysis

Samples must be prepared before protein purification.
Three common ways to lyse cultured E.coli (BL21) cells:

  • - Freeze-thaw
  • - Lysozyme digestion, followed by DNase digestion or mechanical shearing
  • - Sonication

Freeze-thawLysing by repeatedly freezing and thawing the cells.
With E.coli cells:

  • 1. Freezing: Slowly freeze by submerging in liquid nitrogen.
  • 2. Thawing: Quickly thaw in a 37 water bath.
  • 3. Repeat the above steps approximately 3-5 times.
    * BL21 (DE3) pLysS strain contains lysozyme producing DNA. When lysing this cell through the freeze-thawing method, the inner membrane of the cell becomes damaged, releasing this lysozyme and allowing digestion of outer cellular wall.

Lysozyme Digestion

  • 1. Dissolve the lysozyme in an appropriate amount of buffer and adjust the concentration to 10mg/ml.
        * Add enzyme powder to the buffer and dissolve slowly. Do NOT shake or mix vigorously.
        This step must be conducted on ice.
  • 2. In an appropriate amount of buffer, dissolve the E.coli cell pellet and adjust the final concentration of the
        lysozyme stock solution to 1mg/ml.
  • 3. In a 30 incubator, slowly shake incubate for 30-60 minutes or for 16 hours at 4.
  • 4. The product will be very viscous due to the presence of genomic DNA. In order to reduce the viscosity,
        add DNase or MgCl2.
  • 5. When the viscosity has been reduced, centrifuge for 30 minutes at 4 at 20,000 X g.
        Remove the supernatant and save it for use during the purification stage.
        * Because the optimal temperature for lysozyme is 25-30,
        this experiment may be carried out at this optimal temperature if the protein of interest is stable under heat.

SonicationSonication is by far the most efficient method of the three, but because the vibration from the sonicator can produce heat, it must be used with caution. In order to reduce the heat produced in the sample, the sample must be kept in ice and must be sonicated in several short durations (in seconds).

  • 1. Dissolve cell pellet in lysis buffer and keep on ice (the amount of buffer must be 10 times the weight of the pellet
        and sonication must be conducted in ice).
  • 2. Sonicate according to the following schedule:
        15 seconds on, 30 seconds off, at 60% amplitude, repeat 10 times
  • 3. Maintaining 4, centrifuge for 30 minutes at 15,000rpm and collect the supernatant.

Note: Using the above method, prepare the supernatant of the target protein and start the purification process. If the purification process requires a second purification step, exchange the buffer of the elution sample through dialysis and concentrate with amicon concentrator or centricon to use as an input sample for the second purification process.


Purification Overview

Protein purification is an essential experimental method in biotechnology and there are many different ways to purify a protein.
In order to purify a protein to a target purity level, several purification steps are needed. In order to obtain a successful purification result, it is crucial to determine the most effective purification method that uses minimum purification steps to obtain as much protein as possible. Most purification procedures are in chromatography form.
Appropriate purification method must be selected according to the characteristics of the protein that is being purified. The protein purification method below is a chromatography method commonly used in labs.

Characteristics Purification Method
Ligand specificity Affinity Chromatography
Charge Ion Exchange Chromatography
Size Gel Filtration


Affinity Chromatography

Affinity chromatography is a purification method that uses reactions between specific ligands and proteins to separate and purify the protein of interest.
This method is based on the high capacity and resolution of the target protein and has high specialization.

Affinity chromatography is a purification method based on the chemical structure and biological function of individual proteins and can be used in instances where other purification methods cannot be used.

This method allows for the separation of active proteins from a protein possessing different characteristics in function and can purify proteins from a large crude sample.
With the development of the recombinant DNA technology, the production of abundant amount of proteins was made possible. Subsequently recombinant proteins produced through this technology made chromatographic purification easier, thereby allowing for an easier determination of protein characteristics and protein production.

There have been advances in various expression systems that are used as a general method for producing recombinant proteins.
Most recombinant proteins are in fusion protein form with specific affinity tag on the target protein. The use of such tags simplifies the purification of the recombinant fusion protein through the use of affinity chromatography method.

The type of tags differ according to size and characteristics and includes 6 X His tag, GST (glutathione S-transferase) tag, MBP (maltose-binding protein), and FLAG.
FLAG or 6 X His tags are small-size tags and have the benefit of minimizing the effect on the structure, activity, and characteristics of the recombinant protein. FLAG tag is composed of 8 amino acids and has large immunogenicity. Therefore, the tag must be removed after purifying the recombinant protein during antibody production. On the other hand, 6 X His tags have low immunogenicity and thus do not have a large effect on the protein structure or function and can be used without removing the tag.

GST (glutathione S-transferase) and MBP (maltose-binding protein) are considered as large-size tags. GST is a 26kDa protein and is cloned so that it can be connected to the N-term of the target protein, which is expressed in E.coli (pGEX vector). This fusion protein can be easily purified by using an affinity column fixed with glutathione. After purification, the target protein and GST protein can be separated using a specific protease sequence located between the target and GST proteins.

MBP tag is used in pMAL™ Protein Fusion and Purification System. It clones the target protein DNA at the back of malE DNA that produces MBPs, and is expressed as MBP-fusion protein. This fusion protein can be easily purified using amylase column that specifically combines MBPs. MBP fusion proteins have the added benefit of being soluble as MBP increases solubility. This system also has a protease recognizing sequence, allowing for the separation of target protein and MBP via protease processing.

The various systems discussed above are successfully used in research areas such as molecular immunology, vaccine production, protein-protein or DNA-protein interactions.

- 6 His-tag (polyhistidine) Tag: Ni-NTA column
  • 1. This method exploits the high affinity of 6 histidines in the recombinant 6XHis tagged protein and Ni-NTA Agarose.
  • 2. Ni-NTA Agarose: Composed of nitrilotriacetic acid (NTA), 6% agarose matrix, and tetradentate chelating ligand.
        NTA is bound to Ni2+ a due to 4 functional groups.
  • 3. Native & Denaturing conditions:
    1.  Native condition: Used in order to maintain protein activity following cell lysis when the protein of interest is in the supernatant as soluble protein
    2.  Denaturing condition: Used when the protein is insoluble in pellet form and the protein activity has no affect on the target use of the protein.

Ni-NTA Purification MethodBuffer Preparation: Tris and HEPES as well as sodium phosphate and phosphate-citrate are commonly used as buffer reagents. Concentration of the reagent is normally 20-100mM. The purification efficiency can be increased by adjusting the NaCl concentration since the NaCl concentration is connected to protein solubility and column binding capacity depending on protein characteristics. Therefore, it is crucial to adjust the buffer contents and selecting the appropriate buffer for protein purification.

Here, we will be using the two conditions listed below under native conditions.

  • Condition 
  • Buffer A: 20mM Tris-HCl, pH8.0 / 10mM NaCl
  • Buffer B: 20mM Tris-HCl, pH8.0 / 10mM NaCl / 20mM imidazole
  • Buffer C: 20mM Tris-HCl, pH8.0 / 10mM NaCl / 100mM imidazole
  • Buffer D: 20mM Tris-HCl, pH8.0 / 10mM NaCl / 250mM imidazole
  • Condition 
  • Buffer A: 50mM NaH2PO4, pH8.0 / 300mM NaCl
  • Buffer B: 50mM NaH2PO4, pH8.0 / 300mM NaCl / 20mM imidazole
  • Buffer C: 50mM NaH2PO4, pH8.0 / 300mM NaCl / 100mM imidazole
  • Buffer D: 50mM NaH2PO4, pH8.0 / 300mM NaCl / 250mM imidazole

Preparation of Ni-NTA Column

  • 1. Resuspend Ni-NTA Agarose by tapping several times or by inversion.
  • 2. Add approximately 10ml of resuspended resin in the purification column (2.5cm in diameter) and let the resin
       settle for about 5-10 minutes. Then, carefully remove the supernatant from the resin (can also let it flow out by
       opening the stopcock).
  • 3. Add 3 times the amount of D.W as the column volume and resuspend the resin using light tapping motion.
  • 4. Repeat step 2.
  • 5. Add 3 times the amount of binding buffer (Buffer A) as the column volume and resuspend by lightly tapping the
       resin.
  • 6. Repeat step 2.
        (Steps 5 & 6 can be eliminated when using direct purification method as these steps are similar to the equilibration process of
        the following purification step.)

Ni-NTA Purification Process- Native Condition(In every purification step, buffers and samples can be removed by opening the stopcock and letting them flow out)

  • 1. Equilibration: Flow the binding buffer (buffer A), 3-5 times the column volume, through the column
  • 2. Sample binding: Load the prepared purification sample into the column.
        (Prepare the sample by using native condition buffer and set aside a small amount of the sample for SDS-PAGE
         gel verification (Supernatant))
        Collect the samples that flowed through the column in a tube (Flowthrough)
  • 3. Pour the wash buffer (buffer B) (10 times the column volume) through the column.
        Collect the samples in two 50ml tubes. (Wash 1, Wash 2) (50ml x 2 tubes)
  • 4. Elute the protein by using elution buffer (buffer C, D) via an imidazole gradient.
        First, load buffer C (4 times the column volume) and collect the elution samples in 4 tubes. Next, load buffer D
        (4 times the column volume) into the column and collect the elution sample in 4 separate tubes. (Elution 1-8)
  • 5. Sample the eluents as shown below and check on an SDS-PAGE gel.
        : 10µl sample + 5X2.5µl sample dye → 5 mins of boiling → run SDS-PAGE gel

Note: Eluted fraction must be stored at 4 or at -20 (must be mixed with glycerol). When storing for a long period of time, protease inhibitor must be added.Ni-NTA Purification Procedure- Denaturing ConditionIn some cases, recombinant protein expression in an E.coli expression system forms insoluble aggregates. This is called an inclusion body. Such inclusion body proteins can be made soluble by using denaturants such as 6M GuHCl or 8M urea. In denaturing conditions, the 6X His tag of a protein is completely exposed and binds to the Ni-NTA matrix allowing for an effective purification. However, it is sometimes necessary to repeat protein renaturing and refolding processes as the denatured protein possesses characteristics that are different from the native conditions.

The buffers used during Ni-NTA purification under denaturing conditions are as follows.

Denaturing Buffer A: 50mM NaH2PO4, pH8.0 / 300mM NaCl / 8M Urea
Denaturing Buffer B: 50mM NaH2PO4, pH8.0 / 300mM NaCl / 8M Urea / 20mM imidazole
Denaturing Buffer C: 50mM NaH2PO4, pH8.0 / 300mM NaCl / 8M Urea / 100mM imidazole
Denaturing Buffer D: 50mM NaH2PO4, pH8.0 / 300mM NaCl / 8M Urea / 250mM imidazole

  • 1. Prepare samples for purification under denaturing conditions: Add 40ml denaturing buffer A and denature by
        stirring in room temperature for 1 hour (when the protein that needs to be purified exists in an inclusion body
        form in the cell pellet after undergoing sonication and centrifugation processes).
  • 2. Following centrifugation of the sample for 30 minutes at 15,000rpm, transfer the supernatant into a new tube.
        (supernatant)
  • 3. Prepare Ni-NTA Column (Same as above).
  • 4. Equilibration: Flow binding buffer (Denaturing buffer A), 3-5 times the column volume, through the column
  • 5. Sample binding: Load the prepared sample into the column.
        (Prepare the sample by using denature condition buffer and set aside a small amount of the sample for
        SDS-PAGE gel verification (Supernatant))
        Collect the samples that flowed through the column at the bottom of the burette (Flowthrough)
  • 6. Pour the wash buffer (Denaturing buffer B), 10 times the column volume, through the column.
        Collect the samples in two 50ml tubes. (Wash 1, Wash 2) (50ml x 2 tubes)
  • 7. Elute the protein by using elution buffer (Denaturing buffer C, D) via an imidazole gradient.
        First, load buffer C, 4 times the column volume, and collect the elution samples in 4 tubes. Next, load buffer D,
        4 times the column volume, into the column and collect the elution sample in 4 separate tubes. (Elution 1-8)
  • 8. Sample the eluents as shown below and check on an SDS-PAGE gel.
        : 10µl sample + 5X2.5µl sample dye → 5 mins of boiling → run SDS-PAGE gel

Note: The purification step must be conducted at room temperature since the buffer of the denaturing condition can cause crystallization of 8M urea at low temperatures.

- GST (Glutathione-S-Transferase) Tag: GSH column
  • 1. GST is an affinity chromatography method that uses the affinity between GST of GST fusion protein and the reduced form of Glutathione (GSH). GST is a protein that exists as a dimer and binds with GSH when loaded with GST fusion protein. The GST fusion protein is eluted when flowing elution buffer containing GSH through the column since the GST binds with the highly concentrated GSH in the buffer instead of the GSH in the column.
    This method is only available under non-denaturing conditions because this method exploits the characteristics of GST protein.
  • 2. After purification, the target protein can be separated from GST by using site-specific protease.

GST Fusion Protein Purification MethodBuffer Preparation: purification of the GST fusion protein, two types of buffer are used.

  • Condition 
  • Buffer A: 20mM Tris-Cl, pH8.0 / 10mM NaCl / 1mM EDTA
  • Buffer B: 50mM Tris, pH 8.0 / 10mM GSH
  • Condition 
  • Buffer A: 1XPBS (140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, pH7.4)
  • Buffer B: 50mM Tris, pH 8.0 / 10mM GSH

GST Fusion Proteins – GSH Column Purification Method

  • 1. Prepare a column. (Glutathione sepharose 4 Fast Flow)
  • 2. Equilibration: Flow binding buffer (buffer A condition), 3-4 times the column volume, through the column
  • 3. Sample binding: Load the prepared sample into the column.
        (Prepare the sample by using binding buffer and set aside a small amount of the sample for SDS-PAGE gel
        verification (Supernatant))
        Collect the samples that flowed through the column. (Flowthrough)
  • 4. Pour the wash buffer (buffer A), 10 times the column volume, through the column.
        Collect the samples in two 50ml tubes. (Wash 1, Wash 2) (50ml x 2 tubes)
  • 5. Elute the protein by using elution buffer (buffer B) via an GSH concentration gradient.
        Load the buffer B, equal volume with the column volume, and collect the elution samples.
        Repeat the above steps 4 times. (Elution 1-5)
  • 6. Sample the eluents as shown below and check on an SDS-PAGE gel.
        : 10µl sample + 5X2.5µl sample dye → 5 mins of boiling → run SDS-PAGE gel


Ion Exchange Chromatography

Ion exchange Chromatography is a commonly used protein separation/purification method that exploits the high resolution and capacity of ion exchange.

Proteins have a net charge at a specific pH. This charge is based on the degree of positive or negative charge of an exposed amino acid. For example, Arginine, Lysine, and Histidine expresses positive charge at neural pH and glutamic acid and aspartic acid expresses negative charge.
Depending on these charges, proteins express a net charge. When the positive and negative charges of a protein have equal value at a certain pH level, the protein displays a net charge of 0. This pH value is called an isoelectric point (pI).

In order to maximize the separation abilities of the ion exchange chromatography and select the appropriate exchanger, the pI value must be taken into consideration.
For example, if a protein possesses a negative charge at a certain pH, this protein will bind to an anion exchanger that displays a positive value, or if it displays a positive charge, it will bind to a cation exchanger that displays a negative charge.

The ion exchanger is composed of insoluble matrix in which the charged group is covalently bonded. Polystyrene, polyacrylate, dextran (Sephadex), agarose (Sepharose CL-6B), and cellulose (DEAE Sephacel) are used as the matrix.

Charged groups are the basic characteristics of an ion exchanger. The strength and type of the ion exchanger is determined by the type of group; the capacity is determined by the overall number and usefulness of the charged group. The ion exchangers are divided into weak and strong ion exchangers according to the charged groups. Strong and weak forces are defined as the variable ionization levels according to the pH, not the binding strength. Strong ion exchangers are completely ionized in a wide pH range and weak ion exchangers are not.
Commonly used weak ion exchangers are DEAE (diethylaminoethyl), CM (carboxymethyl); strong ion exchangers include Q (quarternary ammonium) and S (sulfonate).

The functional groups of ion exchangers are listed below:

Anion exchangers Functional group
Diethylaminoethyl (DEAE) weak -O-CH2-CH2-N+H(CH2CH3)2
Diethylaminopropyl (ANX) weak -O-CH2CHOHCH2N+H(CH2CH3)2
Quaternary ammonium (Q) Strong -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3
Cation exchangers Functional group
Carboxymethyl (CM) weak -O-CH2-COO-
Sulphopropyl(SP) Strong -O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3-
Methyl sulphonate (S) Strong -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3-

Sepharose Fast Flow Ion ExchangerThe contents below discuss ion exchange columns using DEAE sepharoseTM Fast Flow and CM sepharoseTM Fast Flow.
Sepharose Fast Flow ion exchangers are composed of 90µm high cross-linked 60% agarose beads.
Due to its physical and chemical stability, it can be used to achieve high flow rates commonly required in labs. Moreover, Sepharose Fast Flow is compatible with both strong exchangers (Q, SP) and weak exchangers (DEAE, CM).

- DEAE Sepharose

Prepare the buffer as follows:

Buffer A: 20mM Tris, pH 8.0 / 1mM EDTA
Buffer B: 20mM Tris, pH 8.0 / 1mM EDTA / 100mM NaCl
Buffer C: 20mM Tris, pH 8.0 / 1mM EDTA / 200mM NaCl
Buffer D: 20mM Tris, pH 8.0 / 1mM EDTA / 300mM NaCl
Buffer E: 20mM Tris, pH 8.0 / 1mM EDTA / 500mM NaCl
Buffer F: 20mM Tris, pH 8.0 / 1mM EDTA / 1M NaCl

DEAE Sepharose Column Purification Method

  • 1. Column preparation: DEAE sepharose™ Fast Flow
  • 2. Equilibration: Flow binding buffer (buffer A), 3-5 times the column volume, through the column.
        Sample binding: Load the prepared sample into the column. (Prepare the sample using buffer A and leave
        a small amount of sample for SDS-PAGE gel (Supernatant)).
        Collect the samples that passed through the column in a tube. (Flowthrough)
  • 3. Using the prepared buffer A-F, elute the proteins in NaCl concentration gradient.
        First, add buffer A, twice the column volume, and collect the buffer that passed through the column in
        2 separate tubes. Next, add buffers B, C, D, E, F in this order as instructed for buffer A. Collect all the buffers
        in individual tubes. (Elution 1-12)
  • 7. Sample the eluents as shown below and check on an SDS-PAGE gel.
        : 10µl sample + 5X2.5µl sample dye → 5 mins of boiling → run SDS-PAGE gel
- CM Sepharose

Prepare buffer samples as follows.

Buffer A: 20mM NaH2PO4, pH 6.0 / 1mM EDTA
Buffer B: 20mM NaH2PO4, pH 6.0 / 1mM EDTA / 100mM NaCl
Buffer C: 20mM NaH2PO4, pH 6.0 / 1mM EDTA / 200mM NaCl
Buffer D: 20mM NaH2PO4, pH 6.0 / 1mM EDTA / 300mM NaCl
Buffer E: 20mM NaH2PO4, pH 6.0 / 1mM EDTA / 500mM NaCl
Buffer F: 20mM NaH2PO4, pH 6.0 / 1mM EDTA / 1M NaCl

CM Sepharose Column Purification Method

  • 1. Column preparation: CM sepharose™ Fast Flow
  • 4. Equilibration: Flow binding buffer (buffer A), 3-5 times the column volume, through the column.
        Sample binding: Load the prepared sample into the column. (Prepare the sample using buffer A and leave a
        small amount of sample for SDS-PAGE gel verification (Supernatant)).
        Collect the samples that passed through the column in a tube. (Flowthrough)
  • 5. Using buffers A-F, elute the proteins via a NaCl concentration gradient.
        First, add buffer A, twice the column volume, and collect the buffer that passed through the column in
        2 separate tubes. Next, add buffers B, C, D, E, F in this order as instructed for buffer A. Collect the buffers
        in individual tubes. (Elution 1-12)
  • 8. Sample the eluents as shown below and check on an SDS-PAGE gel.
        : 10µl sample + 5X2.5µl sample dye → 5 mins of boiling → run SDS-PAGE gel


Gel Filtration

- Gel Filtration Overview

Gel filtration is a purification method that exploits the separation that occurs due to differences in protein size during flow through a gel filtration medium. The gel filtration method is different from ion-exchange and affinity chromatography in that the content of the buffer does not directly affect the separation degree of the protein because the protein does not bind to a chromatography medium. Therefore, this purification process can be conducted in relation to the purification and analysis of the following steps. Furthermore, this method can be used to separate proteins under the presence of co-factors, detergents, urea, guanidine, and hydrochloride. Thus, this method can be effectively used for separating proteins that are sensitive to changes in pH levels, ionic strength, and co-factor concentrations.

Gel filtration medium is in a packed bed form in a column. The medium is composed of spherical particles in a porous matrix. The selection of the medium depends on its chemical and physical stability, inertness (lack of reactivity and adsorptive characteristics).

After packing the bed, the buffer is flowed through the column for equilibration in the space between particles and pore of the matrix. The outer part of the particle is the mobile phase and the inner part of the matrix is the stationary phase. When loading the protein sample, the bigger proteins cannot penetrate the small openings inside the matrix and are dispersed through the mobile phase. The smaller proteins penetrate the stationary phase and stay in the column longer, taking longer to elute.

There are two different gel filtration methods that are generally used, but gel filtration usually refers to high resolution fractionation.

Group SeparationThis is a group separation method that quickly removes small molecule groups from large molecule groups by changing buffers. Small molecules such as excess salt or free labels are easily separated through this method. The group separation method is used during protein purification processes such as desalting or buffer exchange.High Resolution FractionationThis method is used for separating proteins according to differences in size. The purpose of high resolution fractionation is to separate out one or more components. Better results can be obtained by first removing similar sized proteins using a separate purification process. Due to this step, this method is normally the last method used during a purification process. Unlike any other purification methods, this method allows for monomer separation.

Gel filtration media must be selected according to the purpose of the experiment, protein of interest, and resolution that optimizes protein separation. There are three types of gel filtration media: Superdex, Sephacryl, and Superose.

Types Characteristics Components
Superdex High level of analysis, fast analysis rate, high recovery rate Covalent bond complex of dextran and high cross-linked agarose: high physical and chemical stability. Viscous eluents such as 6-8M urea can be used.
Sephacryl High level of analysis, fast analysis rate, high recovery rate. Large scale analysis possible. Cross-linked product of allyl dextran and N,N’-methylene bisacrylamide. High stability due to hydrophilic matrix. Viscous eluents such as 6-8M urea can be used.
Superose Wide fractionation range, inappropriate for large scale analysis. Highly cross-linked porous agarose particles, Viscous eluents such as 6-8M urea can be used.


Here, we will be discussing techniques using Sephacryl S100 HR, S300 HR, which are widely used in labs.

Buffer used: 20mM HEPES, pH 7.5 / 150mM NaCl / 1mM EDTA

- Sephacryl Column Purification Method
  • (1) Sample preparation: Concentrate sample to 0.5-4% volume of the entire column. Filter the sample in order to
         remove any particles (0.22-0.45µm filter).
  • (2) Column preparation: Pack the column with sephacryl S 100 HR, S 300 HR.
    2.5cm column diameter, and 100cm in height. (Approximately 500ml)
  • (3) Equilibration: Flow the buffer (twice the volume of that of the column) through the column at a rate of 30cm/h.
  • (4) Sample binding: Load the prepared sample into the column. (In order for verification using SDS-PAGE gel,
         leave a small amount of sample (input)).
  • (5) Obtain protein fraction using a buffer. Conduct fractionation with a pump using the settings below.
         Setting: 1 fraction: 0.5ml/min x 10 mins
    Elute with 1 column volume of buffer
         For best analysis, load with sample (1% column volume) and decrease the flow rate to 15cm/h.
  • (6) Sample the input as well as the fraction sample of interest as directed below and verify on SDS-PAGE.
         : 10µl sample + 5X2.5µl sample dye → boil for 5 mins → run SDS-PAGE gel