3.2 Purification
of Recombinant
Protein
The pAE vector used allows the expression of a recombinant protein with a 6ÂHis fused to its N-terminal. Thus, proteins can be
purified by immobilized metal affinity chromatography (IMAC).
Usually, we use divalent nickel as the metal.
1. Fill a column with chelating Sepharose and wait for resin sedimentation. Afterward, wash the column with 3–5 volumes of
water to remove ethanol used as preservative.
2. Charge the column with 3–5 volumes of nickel solution and
incubate for 30 min. Wash with 3–5 volumes of water.
3. Equilibrate the column with binding buffer.
4. Add protein extract to column.
5. For soluble proteins, wash the column with ten volumes of
wash buffer containing 20 mM imidazole.
6. Wash the column with five volumes of wash buffer containing
40–100 mM imidazole.
7. Elute the bound protein with 3–5 column volumes of elution
buffer. Collect 1 mL fractions in microtubes.
8. In case of insoluble proteins, do on-column refolding by gradually removing the urea. Pass on-column five volumes of binding buffer containing from 8 to 0 M urea. After urea
elimination, proceed starting at step 7.
9. Evaluate the purified proteins using 12% SDS-PAGE. This
percentage is typically used for proteins from 20 to 60 kDa.
Mix 3.3 mL of water, 4 mL of 30% acrylamide mix, and 2.5 mL
of resolving gel buffer. Add 100 μL of SDS, 100 μL of 10%
ammonium persulfate, and 10 μL of TEMED; homogenize
and pour the mixture into a gel cassette. Overlay the gel with
isopropanol until complete polymerization. Prepare 5 mL
stacking gel by mixing 3.4 mL of water, 830 μL of acrylamide
mixture, and 630 μL of stacking gel buffer. Add 50 μL of SDS,
50 μL of 10% ammonium persulfate, and 5 μL of TEMED.
Insert the gel comb carefully. Add 4 μL of 5Â SDS-PAGE
buffer to 16 μL of protein aliquots, and heat the samples for
5 min at 96
C. When running the gel, include protein molecular weight standards. Set the amperage to 25 mA per gel, and
run the gel until the dye reaches the bottom of the gel.
10. Remove the polyacrylamide gel from the glass, and place it in a
suitable container with a lid; stain the gel with Coomassie blue
solution for 30 min or overnight with shaking. Remove the
excess dye using the destaining solution with shaking. Change
the solution until visualization of the protein bands. Figure 1
shows the purification process steps.
178
Aline F. Teixeira and Ana L. T. O. Nascimento
of Recombinant
Protein
The pAE vector used allows the expression of a recombinant protein with a 6ÂHis fused to its N-terminal. Thus, proteins can be
purified by immobilized metal affinity chromatography (IMAC).
Usually, we use divalent nickel as the metal.
1. Fill a column with chelating Sepharose and wait for resin sedimentation. Afterward, wash the column with 3–5 volumes of
water to remove ethanol used as preservative.
2. Charge the column with 3–5 volumes of nickel solution and
incubate for 30 min. Wash with 3–5 volumes of water.
3. Equilibrate the column with binding buffer.
4. Add protein extract to column.
5. For soluble proteins, wash the column with ten volumes of
wash buffer containing 20 mM imidazole.
6. Wash the column with five volumes of wash buffer containing
40–100 mM imidazole.
7. Elute the bound protein with 3–5 column volumes of elution
buffer. Collect 1 mL fractions in microtubes.
8. In case of insoluble proteins, do on-column refolding by gradually removing the urea. Pass on-column five volumes of binding buffer containing from 8 to 0 M urea. After urea
elimination, proceed starting at step 7.
9. Evaluate the purified proteins using 12% SDS-PAGE. This
percentage is typically used for proteins from 20 to 60 kDa.
Mix 3.3 mL of water, 4 mL of 30% acrylamide mix, and 2.5 mL
of resolving gel buffer. Add 100 μL of SDS, 100 μL of 10%
ammonium persulfate, and 10 μL of TEMED; homogenize
and pour the mixture into a gel cassette. Overlay the gel with
isopropanol until complete polymerization. Prepare 5 mL
stacking gel by mixing 3.4 mL of water, 830 μL of acrylamide
mixture, and 630 μL of stacking gel buffer. Add 50 μL of SDS,
50 μL of 10% ammonium persulfate, and 5 μL of TEMED.
Insert the gel comb carefully. Add 4 μL of 5Â SDS-PAGE
buffer to 16 μL of protein aliquots, and heat the samples for
5 min at 96
C. When running the gel, include protein molecular weight standards. Set the amperage to 25 mA per gel, and
run the gel until the dye reaches the bottom of the gel.
10. Remove the polyacrylamide gel from the glass, and place it in a
suitable container with a lid; stain the gel with Coomassie blue
solution for 30 min or overnight with shaking. Remove the
excess dye using the destaining solution with shaking. Change
the solution until visualization of the protein bands. Figure 1
shows the purification process steps.
178
Aline F. Teixeira and Ana L. T. O. Nascimento