Purification Table Judgement and an 80 kDa/160 kDa Mass Discrepancy
A 500 mg sample of crude lysate from an E. coli strain expressing an N-terminally His6-tagged dehydrogenase contains 25,000 U of activity. It is taken through three steps, with assays after each: a 40–60% ammonium sulfate cut (120 mg protein, 18,000 U), DEAE-Sepharose at pH 8.0 (18 mg, 12,600 U) and Ni-NTA eluted with 250 mM imidazole (4.5 mg, 9,000 U). Analytical gel filtration of the final pool on Superdex 200 in 20 mM Tris, 150 mM NaCl gives a single peak at an apparent mass of 160 kDa. SDS-PAGE of the same pool under reducing conditions gives one band at 80 kDa; without 2-mercaptoethanol the band runs at 160 kDa. The protein is needed for a structural study requiring 5 mg at ≥95% purity, and only one 500 mg lysate batch is available.
1. Construct the purification table (specific activity, fold purification, yield, step yield) and identify which step contributes least per unit of activity lost.
Read the worked answer
Specific activity = total activity/total protein; fold = SA at that step/SA of lysate; yield = activity at that step/25,000 U.
| Step | Protein (mg) | Activity (U) | SA (U/mg) | Fold | Yield | Step yield |
|---|---|---|---|---|---|---|
| Lysate | 500 | 25,000 | 50 | 1 | 100% | - |
| (NH₄)₂SO₄ | 120 | 18,000 | 150 | 3.0 | 72% | 72% |
| DEAE | 18 | 12,600 | 700 | 14 | 50% | 70% |
| Ni-NTA | 4.5 | 9,000 | 2,000 | 40 | 36% | 71% |
The DEAE step gives 4.7-fold purification at 70% step yield, while Ni-NTA gives only 2.9-fold at 71% step yield. Judged as purification per unit of activity lost, DEAE is the weaker and more expensive step - it removes protein but is doing work that the affinity step would do far better.
2. Recommend a change in step order, and calculate how much crude lysate protein would be needed to deliver 5 mg of the final protein.
Read the worked answer
The His6 tag makes Ni-NTA an affinity step that should come as early as possible, ideally directly after clarification (a rapid salt cut may precede it to remove lipids and nucleic acid). Placing DEAE before Ni-NTA costs 30% of the activity for purity that Ni-NTA would supply anyway. Moving Ni-NTA to position two should give a similar or better final specific activity with a substantially higher overall yield, and it removes a dialysis/buffer-exchange step between DEAE and Ni-NTA.
Scale calculation: the final specific activity is 2,000 U/mg, and the batch yields 4.5 mg from 500 mg of crude protein. For 5 mg:
Crude protein needed = 500 × (5/4.5) = ~556 mg.
So the single 500 mg batch is short by about 0.5 mg (11%) even before any losses on concentration or polishing. Reordering the steps to raise yield is therefore not optional - it is the only way to meet the 5 mg target from this batch.
3. Interpret the mass discrepancy between gel filtration and SDS-PAGE, and give two experiments that would confirm the quaternary structure.
Read the worked answer
Gel filtration reports the mass of the native, folded assembly (160 kDa); SDS-PAGE with 2-mercaptoethanol reports the polypeptide chain (80 kDa). A native mass exactly twice the subunit mass, with the non-reduced gel also at 160 kDa, means the protein is a disulfide-linked homodimer: the chains are held covalently, so they survive SDS unless the disulfide is reduced.
Confirmation experiments:
- Run SDS-PAGE with and without 10 mM DTT or β-mercaptoethanol on the same sample; the reduced lane must shift to 80 kDa and the non-reduced lane stay at 160 kDa.
- Re-run gel filtration in 6 M guanidine-HCl with and without 10 mM DTT. Guanidine alone will not split a disulfide (the peak stays at 160 kDa); adding DTT should collapse it to 80 kDa. Alternatively, mutate the single cysteine to serine and check by SEC whether the dimer disappears and activity is retained.
For the structural study this matters: 5 mg of the dimer corresponds to 2.5 mg of monomer, and any crystallographic or cryo-EM analysis must be planned for the dimer, with the dimer interface treated as part of the biologically relevant unit.