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Selected applications of IMAC purification of recombinant proteins from crude E.coli extract

A. Multiple–parallel purification of mutant forms of a 6 x His-tagged protein using Proteus IMAC spin columns.

Protocol:

4 g E. coli cell pellet were resuspended in 20 ml buffer A and the cells were lysed by sonication before spinning at 40,000 x g. The Proteus spin column was equilibrated in 10 ml Buffer A. Supernatant (2 x 10 ml) was loaded onto the column in a 5 min spin step. The flow-through was passed back over the column to ensure efficient binding. The column was then washed with 2 x 10 ml wash buffer B, before eluting with 2 x 10 ml elution buffer C.

The flow-through and eluted fractions were then run on an SDS-polyacrylamide gel (Fig A1). The above protocol was applied to the wild-type protein and six site-specific mutants in parallel (Fig A2), yielding >95% pure protein.

• Binding Buffer A: 50 mM sodium phosphate, 0.3 M NaCl, 5 mM β-mercaptoethanol pH 8.0.
• Wash Buffer B: 50 mM sodium phosphate, 0.3 M NaCl , 50 mM imidazole, 5 mM β-mercaptoethanol pH 8.0.
• Elution Buffer C: 50 mM sodium phosphate, 0.3 M NaCl , 250 mM imidazole, 5 mM β-mercaptoethanol pH 8.0.

Fig A1: Lane 1 corresponds to molecular weight markers. Lane 2 corresponds to flow through. Lanes 3 & 4 correspond to the 1st and 2nd final eluate.

Fig A2: Lane 1 corresponds to molecular weight markers. Lane 2 corresponds to final eluate of the wild type protein. Lanes 3 - 8 correspond to final eluates of 6 mutant His-tagged proteins.

B. Purification of C-terminal His-tagged protein using Proteus IMAC Midi spin columns under native conditions.

E. coli cell pellets were lysed by sonication in Buffer A, pH 7.4 and clarified by centrifugation and subsequent filtration. 10 ml sample was loaded on to the buffer A-pre-equilibrated Proteus IMAC column and spun at 150 g for 30 min. The column was washed with 3 x 10 ml wash buffer B and the target protein was eluted twice with 4 ml elution buffer C. The bind-wash-elute cycle was repeated with a further 10 ml sample to ensure that the performance of the column was unaffected in the second bind-wash-elute cycle when there was no further charging of the column with Ni metal.

The binding capacity of the Proteus IMAC column was unaffected after two consecutive purification runs. The recovered capacity of the column was 18 mg recombinant protein after the first purification cycle and 20 mg recombinant protein after the second purification cycle. This exceeded the stated 10 mg capacity of the column. SDS-PAGE confirms the excellent purity of the target protein (see Fig. B).

• Binding Buffer A: PBS buffer pH 7.4.
• Wash Buffer B: PBS buffer pH 7.4, 1 M NaCl, 20 mM imidazole.
• Elution Buffer C: PBS buffer pH 7.4, 300 mM imidazole.

C. Purification of a His-tagged protein under denaturing conditions using the Proteus IMAC Mini spin columns

Filtered cell extract was equilibrated with 2x 0.65 ml buffer A by centrifuging the spin column at 1,800 g for 1 min. Sample (0.65 ml) was spun through the spin column at 640 g for 6 min. The spin columns were washed 5 times with 0.65 ml wash buffer B and the bound recombinant protein was eluted twice with 2 x 0.65 ml elution buffer C by centrifuging the spin column at 1,800 g for 1 min. Nearly all of the recombinant protein was eluted in the elution step with elution buffer C. Excellent purity was observed in the SDS-polyacrylamide gel (Fig. C). A total of 0.55 mg recombinant protein was recovered from the final eluate.

• Binding Buffer A: 0.1 M sodium phosphate, 10 mM Tris, 10mM imidazole, 8 M Urea pH 8.0
• Wash Buffer B: 0.1 M sodium phosphate, 10 mM Tris, 20 mM imidazole, 8 M Urea pH 8.0
• Elution Buffer C: 0.1 M sodium phosphate, 10 mM Tris, 250 mM imidazole, 8 M Urea pH 8.0

Fig C: SDS-polyacrylamide gel (12.5 %) showing the purity of the His-tagged recombinant protein. Lane 1 corresponds to the molecular weight markers; Lane 2 corresponds to the sample load; Lane 3 corresponds to the flow through; Lanes 4-8 correspond to the wash steps and Lanes 9 & 10 correspond to the 1st and 2nd final eluates.

D. Purification and multiple loading of a membrane-associated His-tagged protein under native conditions using the Proteus MC Midi spin columns

7 g E. coli cell pellet was re-suspended in 60 ml lysis buffer pH 8.0 (+ 0.01 % Triton X-100) and lysed by sonication. The suspension was filtered through a 0.2 µm filter and loaded on to the pre-equilibrated spin column in 3 x 20 ml aliquots. The spin column was washed with 3 x 10 ml wash buffer B and the target protein was eluted with 10 ml elution buffer. The sample flow through, washes and eluate were run on a 10 % SDS-polyacrylamide gel prior to Western blotting. The Western blot (Fig. D) showed no trace of the 50 kDa target protein in the flow through or wash steps, demonstrating an extremely high binding efficiency of the His-tagged target protein to the Proteus metal chelate column.

• Binding Buffer A: 50 mM sodium phosphate, 0.3 M NaCl, 10mM imidazole, 0.01 % Triton X-100 pH 8.0
• Wash Buffer B: 50 mM sodium phosphate, 0.3 M NaCl, 30mM imidazole, 0.01 % Triton X-100 pH 8.0
• Elution Buffer C: 50 mM sodium phosphate, 0.3 M NaCl, 300mM imidazole, 0.01 % Triton X-100 pH 8.0

Fig D: Western blot showing the purity of the membrane associated His-tagged recombinant protein in lane 5 and the absence of target protein in the flow through and washes (lanes 1-4). Lane 1 responds to the flow through; Lanes 2-4 correspond to the wash steps and Lane 5 corresponds to the final eluate.