Protein purification

Purification of a His6-tagged Green Fluorescent Protein (GFP)

5.2 Protein purification

5.2.1 Purification of a His6-tagged Green Fluorescent Protein

(GFP)
Principle
You can add either a N- or C-terminal His6-tag to the protein that you want to express if
you use the RTS pIVEX His6-tag 2nd generation vector set (pIVEX2.3d; pIVEX2.4d, see
Chapter 2.4.2.1) or the RTS E. coli Linear Template Generation Set, His6-tag (see Chapter
2.3.3.1).

These His6-tagged proteins can be purified in one step by immobilized metal affinity
chromatography (IMAC) (Ford, C. F. et al., 1991) on a nickel-nitrilotriacetic acid (Ni-
NTA) column. In a single step, this affinity matrix can purify a protein (starting concen-
tration less than 1% of the total protein) to more than 95% homogeneity.

Nitrilotriacetic acid (NTA) is a tetradentate chelating adsorbent developed by Roche

Diagnostics GmbH. NTA occupies four of six ligand binding sites of the nickel ion, leav-

5
ing two sites free for interaction with the His6-tag. NTA binds metal ions tightly, allowing
use of stringent washes.

Histidine residues on the tag, connected via a short linker to the C- or N-terminus of the
protein, bind to the Ni-ions. The protein can be eluted by competitive displacement with
imidazole.

Note: Since Ni-NTA is not as selective as other affinity chromatography matrices, it may
also bind proteins with exposed patches of histidine, cysteine or tryptophan residues.
Therefore elution conditions must be optimized for each protein. An easy way to opti-
mize conditions is to use an imidazole gradient for elution, rather than a single imidazole
concentration.

Protocol
The following purification protocol is optimized for purification of His6-tagged GFP. If it
is used to purify other proteins, the protocol may have to be modified. For more detailed
information see the manufacturer’s handbook provided with the purification matrix.

Material required

Reagent Vendor
Ni-NTA agarose QIAgen

1 ml column with luer lock on both ends MoBiTec

10 ml luer lock syringe Merck Eurolab

Buffer Composition
Equilibration buffer 20 mM Tris/HCl, 200 mM NaCl; pH 7.5

Washing buffer 20 mM Tris/HCl, 200 mM NaCl, 5 mM imidazole; pH 7.5

Elution buffer 1* 20 mM Tris/HCl, 200 mM NaCl, 20 mM imidazole; pH 7.5

Elution buffer 2* 20 mM Tris/HCl, 200 mM NaCl, 200 mM imidazole; pH 7.5

Elution buffer 3 20 mM Tris/HCl, 200 mM NaCl, 500 mM imidazole; pH 7.5

*The imidazole concentrations of elution buffers 1 and 2 must be optimized for each protein.
164 RTS Application Manual
 Protein purification
Purification of a His6-tagged Green Fluorescent Protein (GFP)

Notes on use of Ni-NTA column:

X We recommend using new Ni-NTA resin for each purification.

X Use only gravity flow for all purification steps.

X Perform all steps at 4°C.

X Before mounting the luer lock syringe on top of the column, make sure that
the column is filled to the top with buffer and that all air bubbles remain above
the resin bed. Remove these air bubbles with a syringe or thin pipette tip.

X When changing the buffer:

XLet the current buffer flow through the column until the buffer reservoir
is nearly empty.
XAdd only a small volume of the new buffer.
XLet that volume flow through the column until the reservoir is nearly
empty again.
XFinally, begin washing the column extensively with the new buffer.

X Make sure that the column does not run dry at any point in the procedure.

Procedure for purification of a His6-tagged protein

Step Action
5
1 X Fill the column with Ni-NTA resin to create a bed volume of 0.6 ml.
X Close the column and mount the luer lock syringe (without plunger) as a buffer
reservoir.
X Equilibrate the column with 10 to 15 bed volumes (6 – 9 ml) of equilibration buffer.

2 X Apply the sample to the column by gravity flow. Keep a small portion of the sample
for assays (in Step 4).
Note: Often, you can apply the contents of the RTS reaction chamber directly to
the column. However, if you see precipitate in the sample that might clog the
column, centrifuge the sample at 10 000 x g for 1 min to remove the precipitate
before applying the sample to the column.

3 X Immediately after the sample has entered the resin, wash the column with 10 bed
volumes (6 ml) washing buffer.

4 X Beginning with the first washes, collect 1 ml fractions of effluent from the column
throughout the entire purification.
X Monitor the progress of the purification by analyzing each fraction by SDS-PAGE,
Western blotting and/or activity assay. Use the unpurified sample as a reference in
these assays.

5 X Elute nonspecifically bound proteins with 10 bed volumes (6 ml) elution buffer 1.
Note: The imidazole concentration in elution buffer 1 must be optimized for each
protein.

6 X Elute specifically bound protein with 10 bed volumes (6 ml) elution buffer 2.
Note: The imidazole concentration in elution buffer 2 must be optimized for each
protein. For GFP with the His6-tag on either end, 200 mM imidazole showed the
best results.

7 X After all specifically bound protein has been eluted from the column, wash the
column with 10 bed volumes (6 ml) elution buffer 3. This will elute all bound
proteins from the column.

Protein analysis and purification 165

 Protein purification
Purification of a His6-tagged Green Fluorescent Protein (GFP)

Typical result
Figure 57 shows an SDS-PAGE assay of the purification of GFP with a C-terminal His6-
tag. The recovery of purified protein was about 96%. Of that total, 94% emerged in the
first two fractions eluted with elution buffer 2 (Figures 57 and 58).

Figure 57: Purification of GFP with a

C-terminal His6-tag: Assay of eluted
fractions by 10% Bis/Tris (MOPS)
SDS-PAGE.
1: Molecular weight standard; 2: Crude
extract; 3: Flow-through; 4: Pooled wash-
ing fractions; 5: Elution 1 with 20 mM imi-
dazole, pooled; 6-8: Elution 2 with 200 mM
imidazole, fractions 1, 2, and 3 respec-
tively.

5 900

800

700

600
GFP [µg]

500

400

300

200

100

0
crude extract flowthrough washing elution 1 elution 2

Figure 58: Recovery of His6-tagged GFP during the purification procedure.

166 RTS Application Manual

 Protein purification
Purification of an MBP fusion protein

5.2.2 Purification of an MBP fusion protein

Principle
To increase the solubility of a protein, express it with an N-terminal MBP fusion. This
fusion can be added with either the RTS E. coli Linear Template Generation Set, MBP
Fusion (see Chapter 2.3.3.4) or the pIVEX-MBP cloning vector (see Chapter 2.4.2.3).

The expressed fusion protein can then be purified in one step by affinity chromatography
on amylose matrices (Maina, C.V. et al., 1988). The maltose binding protein, connected
via a short linker to the N-terminus of the desired protein, binds to the amylose resin.
The protein can be eluted by competitive displacement with maltose. The protein may
also be cleaved with Factor Xa protease (see Chapter 5.3), either while it is still bound to
the column or after it is eluted.

The fusion protein carries an additional N-terminal His6-tag that would allow one-step
purification by Ni-NTA affinity chromatography (see Chapter 5.2.1).

Protocol
The following purification protocol is optimized for the purification of MBP-endogly-

5
cosidase. If it is used to purify other proteins, the protocol may have to be modified. For
more detailed information see the manufacturer’s handbook provided with the purifica-
tion matrix.

Note: The resin can be reused three to five times if it is regenerated according to the
manufacturer’s manual.

Material required

Reagent Vendor
Amylose resin New England Biolabs

1 ml column with luer lock on both ends MoBiTec

Buffer Composition
Equilibration buffer 10 mM Tris-HCl; pH 7.2

Wash buffer 10 mM Tris-HCl, 1 M NaCl; pH 7.2

Elution buffer 10 mM Tris-HCl, 10 mM maltose; pH 7.2

Protein analysis and purification 167

 Protein purification
Purification of an MBP fusion protein

Procedure

Step Action

1 X Perform the following steps at 4°C, using only gravity flow.

2 X Pour the appropriate amount of amylose resin into the column.

Note: In general, 1 ml resin is enough for one RTS 500 E. coli HY reaction. The
binding capacity of amylose resin is approx. 3 mg/ml.
X Equilibrate the column with 8 column volumes of equilibration buffer.

3 X Centrifuge the sample at 10 000 x g for 1 min to remove any precipitated protein
that might clog the column.
X Apply the supernatant to the column by gravity flow. Keep a small portion of the
supernatant for assays (in Step 6).
X Once all the sample has entered the column, shut off the column flow.

4 X Incubate the column (containing the sample) for 15 min to enable optimal binding
between the fusion protein and the amylose resin.

5 X Wash the column with 10 bed volumes of wash buffer.

5
6 X Beginning with the first washes, collect fractions of effluent from the column
throughout the entire purification.
Note: Fraction size should be 1/3 of column volume.
X Monitor the progress of the purification by analyzing each fraction by SDS-PAGE,
Western blotting and/or activity assay. Use the unpurified sample as a reference in
these assays.

7 X Elute bound protein with 2–4 bed volumes of elution buffer.

Note: The fusion protein usually starts to elute within the first 5 fractions. It should
easily be detected by UV absorption at 280 nm or the Bradford protein assay.

Typical result
Figure 59 shows an SDS-PAGE assay of the purification of MBP-endoglycosidase on amy-
lose resin. Staining was done with SimplyBlue Safe Stain (Invitrogen). The additional
bands in lanes 9 and 10 are degradation products of the MBP fusion protein.

Figure 59: Purification of MBP-endoglycosidase:

Assay of eluted fractions by 10% Bis/ Tris (MOPS) SDS-PAGE.
1: Supernatant of reaction solution; 2: Molecular weight standard; 3–5: Flow-through; 6–7: Washing fractions;
8–10: Elution fractions; 10 µl of sample were loaded on each lane.

168 RTS Application Manual

 Protein purification
Purification of an HA-tagged fusion protein

5.2.3 Purification of an HA-tagged protein

Principle
In some cases, you may need an alternative tag. You can use either the RTS E. coli Linear
Template Generation Set, HA-tag (see Chapter 2.3.3.2), or the vectors pIVEX2.5d and
pIVEX2.6d (see Chapter 2.4.2.2) to attach either a C-terminal HA-tag (pIVEX2.5d) or an
N-terminal HA-tag (pIVEX2.6d) to the protein that you want to express.

These HA-tagged proteins can be purified in one step by affinity chromatography on an

Anti-HA Affinity Matrix. The Anti-HA Affinity Matrix is also suitable for:

X Affinity purification of HA-tagged proteins from crude protein extracts.

X Immunoprecipitation of HA-tagged proteins from mammalian, yeast and bacterial

cell extracts.

The purified protein can be analyzed on a Western blot with an Anti-HA antibody.

Protocol

5
The following purification protocol is optimized for the purification of an HA-tagged
GFP mutant. If it is used to purify other proteins, the protocol may have to be modified.
For further details see the pack insert of the Anti-HA Affinity Matrix (Cat. No.
1 815 016).

Material required

Reagent or equipment Vendor Cat. No.

Anti-HA Affinity Matrix Roche Applied Science 1 815 016

HA peptide Roche Applied Science 1 666 975

1 ml column with luer lock on both ends MoBiTec

23-gauge (23 G) needle for adjustment of flow rate

Note: This needle size is suitable for a 0.5–1.0 ml
column. Adjust needle size as needed for larger or
smaller columns.

Buffer* Composition
Equilibration buffer 20 mM Tris-HCl, 0.1 M NaCl, 0.1 mM EDTA; pH 7.5

Wash buffer 20 mM Tris-HCl, 1 M NaCl; pH 7.5

Elution buffer Dissolve HA peptide at 1 mg/ml in equilibration buffer.

Note: Store at –20°C and thaw before use.

Column storage buffer 20 mM Tris, 0.1 M NaCl, 0,1 mM EDTA, 0.09% sodium-azide; pH 7.5

Regeneration buffer 0.1 M Glycine, pH 2.0

* 100 ml of each buffer should be enough to purify several expressed proteins. All buffers
except the elution buffer may be stored at 2°–8°C for up to one month (store the elution
buffer at –20°C). Bring buffers to room temperature before use.

Protein analysis and purification 169

 Protein purification
Purification of an HA-tagged fusion protein

Procedure
A. Preparing the column

Step Action

1 X Perform all the steps of the purification at 4°C unless noted otherwise. Use only
gravity flow to elute the column.

2 X Attach lower cap to bottom of column, and place column on rack or stand above a
collection tube.

3 X Gently invert Anti-HA Affinity Matrix several times to thoroughly resuspend beads.

4 X Pipet desired volume of slurry into column.

Note: We suggest a final settled bead volume of 0.5–1.0 ml.

5 X Replace lower cap with 23 G needle.

6 X Drain column storage buffer into collection tube.

Note: Do not allow column to dry out. Flow rate should be approximately
0.3–0.5 ml/minute. If flow rate is faster, use a smaller gauge needle to adjust the rate.

5
7 X Immediately add 10 bed volumes of equilibration buffer and allow buffer to drip
through column.

B. Loading the column

Step Action

1 X Determine amount of crude protein extract (RTS reaction solution) to be purified.

Notes:
X Optimal sample volume is 1–4 ml for a 1 ml column.
X Amount of total protein in RTS reaction solution is typically 1–5 mg. However,
the total protein concentration of the solution depends upon expression levels
of tagged protein.

2 X Centrifuge the sample at 10 000 x g for 1 min to remove any precipitated protein
that might clog the column.
Note: Column may become fouled if excessive total protein is loaded or insoluble
materials are not completely removed from the RTS reaction solution.
X Apply the supernatant to the equilibrated column by gravity flow.

3 X Collect flow-through in a clean collection tube. Save this crude protein extract
fraction at 4°C.

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 Protein purification
Purification of an HA-tagged fusion protein

C. Eluting proteins

Step Action

1 X Wash column with a minimum of 20 bed volumes of washing buffer at room tem-
perature to remove nonspecifically bound protein.
Note: Read the OD280 of the effluent at the end of this step to verify that the final
wash fractions contain no protein and are close to baseline levels (= wash buffer
alone).
X Save wash fractions if desired.

2 X Immediately replace needle with lower column cap.

X Add 1 bed volume of elution buffer to the column and incubate for 15 minutes at
37°C.
Note: Elutions may be performed at a lower temperature; however this will lower
the yield of purified protein.

3 X Replace the lower cap with the needle and collect the elution fraction in a clean
collection tube (microcentrifuge tube or equivalent).
X Keep samples cold (4°C) until they are analyzed.

5
4 X Repeat Steps 2 and 3 twice.

5 X Read OD280 for each fraction and pool as desired.

Note: The yield of purified protein depends upon expression levels. Typical results
from a 1 ml column range from 2 to 8 nmol of purified protein.

D. Column reuse, regeneration and storage

Step Action

1 X Strip column by running 20 bed volumes of regeneration buffer through the column.
Note: Depending on the protein used, complete regeneration of the matrix may
require up to 160 bed-volumes of regeneration buffer.

2 X Immediately re-equilibrate column with 20 bed volumes of equilibration buffer.

3 X Store column tightly capped at 4°C in 2 bed volumes of column storage buffer.
Note: The affinity matrix may be used at least ten times. Suitability of the column
for more than ten uses should be determined by the user and will depend upon
the tagged protein or cell extract used.

Typical result
Figure 60 shows an SDS-PAGE assay of the purification of an HA-tagged protein on an
anti-HA matrix. The gel was stained with Coomassie Blue.

Figure 60: 10% Bis/Tris (MOPS) SDS-PAGE

illustrating the purification of an HA-tagged
GFP mutant.
1: Molecular weight standard, 10 µl; 2: pIVEX 2.6 GFP
mutant expression, supernatant; 3: Wash fraction;
4: Elution step 1; 5: Elution step 2; 6: Elution step 3;
7: Elution step 4; Samples loaded on the gel were 0.5 µl
pIVEX 2.6 GFP mutant expression supernatant (2), 5 µl
wash fraction (3) and elutions (4, 5, 6, 7).

Protein analysis and purification 171

 Protein purification
Troubleshooting the purification procedures

5.2.4 Troubleshooting the purification procedures

Observation Possible cause Recommendation

His6-tag

Fusion protein His6-tag not present X Check sequence and reading frame.
does not bind X Check for possible internal translation starts (N-terminal
to the column. tags) or premature termination sites (C-terminal tags).

Binding conditions incorrect X Check that the correct buffers and pH have been used.
X Decrease the concentration of imidazole in the binding buffer.
X Ensure that there are no chelating or reducing agents present.
X If reusing a column, make sure it has been regenerated correctly.

Tag may be inaccessible. X Put tag on another part of the protein.

X Purify protein under denaturing conditions (e.g.,sodium
phosphate buffer, pH 8.0 with 8 M urea or 6 M guanidinium
hydrochloride; optionally, add 10-500 mM imidazole to
increase binding specificity).

Tag may be degraded. X Include protease inhibitors and perform purification at 4°C.

5 Protein elutes in
the wash buffer.

Fusion protein
elutes poorly.
Column capacity is exceeded

Wash stringency too high

His6-tag partially hidden

Column may not have been

correctly charged with nickel
X

X
X

X
Apply less fusion protein to column.

Lower the concentration of imidazole or increase pH.

Reduce wash stringency.

Purify under denaturing conditions.

Repeat column preparation steps.

ions.

Elution conditions too mild X Increase concentration of imidazole in the elution buffer
(>400 mM).
X Use gradient with increasing concentrations of imidazole.
X Carefully lower pH to create more stringent elution conditions.
Note: Do not use a pH below pH 3.5 because low pHs will
strip metal ions off the column.

Fusion protein may be X Purify protein at room temperature or at 4°C: Fill the column
precipitating. with elution buffer, incubate overnight and elute the protein
with elution buffer the following day.
X Add solubilizing reagents, e.g., 2 M NaCl, 50 mM CHAPS,
50% glycerol, 8 M urea, 6 M guanidine hydrochloride,
0.1-2% Tween 20, 0.1–2% Triton X-100.
Note: Triton X-100 has a high absorbance at 280 nm, and
cannot be removed by buffer exchange procedures.
X Add reducing agents such as 2-mercaptoethanol to help
solubilization.
X Perform binding and elution in batch format to avoid high
local concentrations of protein.

Protein elutes with Binding and wash conditions X Use more stringent binding or washing conditions.
contaminants. not stringent enough

Contaminants associated with X Increase salt and/or detergent concentration or add glycerol
tagged protein. to wash buffer to disrupt nonspecific interactions.

Column too large X Reduce amount of Ni-NTA matrix used.

Contaminants are truncated X Check sequence for possible internal translation starts (C-ter-
forms of tagged protein. minal tags) or premature termination sites (N-terminal tags).

Discoloration Nickel ions are removed X Ensure that there are no chelating agents (which turn resin
of resin or reduced. white) or reducing agents (which turn resin brown) present
in the buffers.

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 Protein purification
Troubleshooting the purification procedures

HA-tag

Little or no HA-tag- Tagged protein is degraded. X Include protease inhibitors and perform purification at 4°C.
ged protein is
eluted. Tagged protein not fully X If working at less than 37°C, increase temperature.
eluted X Alternatively, increase time and/or volume of elution buffer.
X Try batch mixing of peptide solution with matrix.

No or low expression of X Load larger volume of extract.

tagged protein X Run column several times.

Large quantities of Column is overloaded X Decrease amount of loaded protein extract.

tagged protein pre-
sent in the flow- Column not regenerated X Regenerate column before repurifying protein.
through sample after last use

Tagged protein Protease activity during X Increase protease inhibitors in protein sample.
appears degraded procedure X Perform all steps at a lower temperature.
(smear or multiple
lower molecular
weight bands seen
on Western blot).

MBP Fusion

Little or no MBP-
tagged protein is
eluted.
Tagged protein not fully
eluted
X
X
Try higher concentration of maltose (up to 500 mM).
Try batch mixing of solution with matrix.
5

Protein analysis and purification 173