This purification protocol was optimized for a poorly expressing protein in E. coli under denaturing conditions.
Material recommended: Gradient maker pump system (e.g. BioRad BioLogic system, Pharmacia gradiFrac or HPLC, etc.)
1) Prepare several litters of bacterail culture: Make a pre-culture over-night (for example 500 ml) without IPTG induction.
2) Inoculate the desired volume of fresh culture medium by a 1/10 dilution of the pre-culture. Incubate at 37°C for around 1 hour. Add 0.5 to 2 mM IPTG. Let grow for 4 to 8 hours.
3) Collect cells by centrifugation.
4) Ressuspend the cells in 8 M urea, 1M NaCl, 10% glycerol, pH 8 (sonication buffer). Sonicate around 8 to 10 min in ice with for example 6 sec impulses at 15 to 20 amplitude.
Note: For a weakly expressed protein a very concentrate lysate is the best choice, so whatever the culture volume, the lysate volume should not exceed around 30-35 ml.
5) Make the best clarification you can! Specially for concentrate lysates, to minimize the risks of column occlusion. For example, a 45 min and 11500 rpm centrifugation will do the job.
6) Harvest the clear supernatant and add 0.5% Triton X-100 (reduced if possible) and 5-10 mM Imidazol. remove bubbles by degasing.
Note 1: For a weakly expressed protein, non-specific interactions are a serious problem. Thus, a quite stringent condition should be used. The condition described here is far from the maximum allowed (but gives good results already), so some improvements can be obtained by increasing NaCl or glycerol for example.
Note 2: NaCl reduces ionic interactions, glycerol and Triton X-100 reduce hydrophobic interactions, Imidazol reduces weak and endogenous histidine interactions.
7) Use a 1 ml Ni column (HisTrap from Pharmacia for example). Equilibrate it with sonication buffer (8M urea, 1M NaCl, etc) at pH 8.
8) Pass the lysate throw the column. Save the Flow-throw. Wash the columns with around 30 ml of urea buffer pH 8 (sonication).
Note: Elution with pH drop is the best choice under denaturing conditions. It allows a subsequent purification if needed by simply raising pH to 8.
9) Begin elution by increasing elution buffer (sonication buffer adjusted to pH 4.95) proportion with gradual increase or isocratic step. A gradient is better for a first run. Contaminants will eluate first as pH falls.
Note: Elution buffer pH is highly crucial! adjust it carefully to 4.95. A small 0.5 pH diference inluence the elution time of the tagged protein.
In principle, a step to 90% elution buffer amount can be performed to eliminate most of contaminants. Let it flow for around 20 ml. Jump to next step at 95% for about 10 ml.
10) Elute by a 100% elution buffer step (pH 4.95) for around 10 ml.
Note: These values are indicative. In a first run, a gradient with holds should be performed to find adapted conditions.
11) Analyse fractions by a SDS-PAGE and Coomassie staining.
Reagents usage and compatibility with his-tag purification
Max. conc. allowed
Tris buffer, NOT recommended
NaCl 2 M prevents ionic interactions
Imidazol histidine competitor
Glycerol 30 % prevents hydrophobic interactions
Triton X-100, NP-40, CHAPS 2 % detergent, prevents hidrophobic interactions
Urea 8 M denaturing agent