3.3 Protein
Purification from
Bacterial Lysates
The protocols below are applicable to the purification of N- and
C-terminal recombinant fragments.
3.3.1 Cell Lysis
1. Thaw cell pellet on ice and add 5–10 mL ice-cold lysis buffer
per g pellet to the thawed cell pellet.
2. Solubilize the pellet until a homogeneous solution is reached.
A magnetic stirrer may be used for effective solubilization.
3. Lyse cells by using a cell disrupter at 4
C, applying a pressure
of 26 kpsi, and repeat this step once.
4. Pellet cell debris by centrifugation for 30 min, at 4
C and
30,000 Â g (see Note 5).
3.3.2 His-Trap
Purification
1. Filter cell lysate using a syringe and a 0.45 μm syringe filter.
Subsequently, dilute it 2/1 in wash buffer. Maintain the lysate
sample at 0–4
C during the entire purification process by
performing the next steps in a refrigerator and keeping samples
on ice.
2. Measure the pH of the sample and adjust it to pH 7.4 with 1 M
HCl or NaOH (see Note 6).
3. Prepare a 5-mL His-trap FF column by washing it with 3 column volumes (CV) deionized water and equilibrating it with
5 CV wash buffer. This can be performed with either a peristaltic pump with a flow rate of 3.5 mL/min or manually with a
syringe.
4. Load the filtered and diluted lysate sample onto the column
and remove nonbinding residues by washing it with 20 CV
wash buffer.
5. Elute the His-tag protein with 5 CV elution buffer and collect
the flow-through in 1.5 mL fractions in 2 mL reaction tubes.
6. Measure the protein concentration of each fraction on a spectrophotometer at 280 nm. Then verify purity and molecular
weight of the protein with UPLC and LCMS, respectively.
Pool pure fractions to target a purity of >85–90% from
UPLC (see Note 7).
7. Concentrate the combined sample using centrifugal filters with
a MWCO below the molecular weight of the respective
protein.
3.3.3 FPLC Purification
If the protein sample does not meet target purity of >85–90% after
the His-trap purification, a size-exclusion (SEC) purification step
can be performed using an FPLC. A flow rate of 1 mL/min at 4
C
is recommended.
1. Equilibrate the column with 5 CV wash buffer.
204
Christin Kossmann et al.
Purification from
Bacterial Lysates
The protocols below are applicable to the purification of N- and
C-terminal recombinant fragments.
3.3.1 Cell Lysis
1. Thaw cell pellet on ice and add 5–10 mL ice-cold lysis buffer
per g pellet to the thawed cell pellet.
2. Solubilize the pellet until a homogeneous solution is reached.
A magnetic stirrer may be used for effective solubilization.
3. Lyse cells by using a cell disrupter at 4
C, applying a pressure
of 26 kpsi, and repeat this step once.
4. Pellet cell debris by centrifugation for 30 min, at 4
C and
30,000 Â g (see Note 5).
3.3.2 His-Trap
Purification
1. Filter cell lysate using a syringe and a 0.45 μm syringe filter.
Subsequently, dilute it 2/1 in wash buffer. Maintain the lysate
sample at 0–4
C during the entire purification process by
performing the next steps in a refrigerator and keeping samples
on ice.
2. Measure the pH of the sample and adjust it to pH 7.4 with 1 M
HCl or NaOH (see Note 6).
3. Prepare a 5-mL His-trap FF column by washing it with 3 column volumes (CV) deionized water and equilibrating it with
5 CV wash buffer. This can be performed with either a peristaltic pump with a flow rate of 3.5 mL/min or manually with a
syringe.
4. Load the filtered and diluted lysate sample onto the column
and remove nonbinding residues by washing it with 20 CV
wash buffer.
5. Elute the His-tag protein with 5 CV elution buffer and collect
the flow-through in 1.5 mL fractions in 2 mL reaction tubes.
6. Measure the protein concentration of each fraction on a spectrophotometer at 280 nm. Then verify purity and molecular
weight of the protein with UPLC and LCMS, respectively.
Pool pure fractions to target a purity of >85–90% from
UPLC (see Note 7).
7. Concentrate the combined sample using centrifugal filters with
a MWCO below the molecular weight of the respective
protein.
3.3.3 FPLC Purification
If the protein sample does not meet target purity of >85–90% after
the His-trap purification, a size-exclusion (SEC) purification step
can be performed using an FPLC. A flow rate of 1 mL/min at 4
C
is recommended.
1. Equilibrate the column with 5 CV wash buffer.
204
Christin Kossmann et al.