Every biochemist knows that purification of proteins should be as fast as possible because proteins are prone to non-specific cleavage or structure instability due to thermal energy in the environment. In the following post(s) I will detail how I broke my own record of protein purification time.
I am working on this protein for biochemical analysis, nothing crystallographic, so any purity from 80% and northward was good for me. This protein was purified in the past and generally I wanted to utilize only two chromatographic steps. While working on the bench I’ve logged all observations (quantitative and qualitative) in my BioKM account which assured me that my data will not be lost while on the bench. Learn how BioKM can improve your research at the lab here.
OK, so we need to get that recombinant protein out of the cell mass. We use the old faithful and tough French press from Thermo which is getting a collector’s piece as Thermo stopped producing this really effective piece of equipment.
7:45am – I’ve submerged the French press cylinder and etc under a layer of ice in an ice box. While the French gets cold, I poped the pellet (20 grams in a 50ml tube) out of the -80°C freezer and overlaid it with binding buffer (essentially ~0.5M NaCl, Tris buffer some Triton X-100 to help with membrane disruption) supplemented with protease inhibitors and DNAse I. Don’t forget those!
We have a rotating shaker so I’ve put that stuff on the maximum speed – from time to time I’ve do manual vortexing of the tube to aid in resuspension. When ~30ml of the stuff was already in liquid state I’ve mounted the French press apparatus and started performing one pass of lysis. The rest was kept on the rotating shaker (I’ve also added some binding buffer to aid in the resuspension).
8:30 – After performing two passes of lysis at 1500 PSI (high pressure mode) I’ve moved to lysing the rest of the pellet while the lysed stuff I’ve buried under lots of ice.
8:45 – Once both were lysed I moved the suspensions onto ~37ml ultracentrifuge tubes. Watch-out – these babies can have deformations and cracks. You don’t want to be the unlucky student who messed up with the ultra! I have weighted all tubes so that each pair will contain the same volume of bacterial suspension so it will be easier to polish their balance with ease using binding buffer (NOT water!). Balance between each pair should no exceed 0.02gr!
9:00 – Running the old ultracentrifuge at 123,000g for 1.5hr (usually we use 203,000g for 45’ but I wanted as clean as possible supernatant). Don’t leave it alone while accelerating!
9:10-10:40 – While the ultra runs, I’ve cleaned the French from the lysis and let it dry in the chemical hood. I also mounted the Bio-Rad plastic column with ~3ml of Ni-NTA beads (Novagen) which were immediately washed with 10CV of water and binding buffer (equilibration step). Updated my ELN and answered a few emails.
Affinity chromatography – Ni-NTA column
10:40 – The pellet looked ok (brown-dark colored) so there are no inclusion bodies. Immediately the supernatant was loaded on the column and rotated at room temp for 5 minutes (batch binding).
10:55 – Finished extensive wash steps (total 40CV with three different wash buffers). Start elution at 1ml step, recording each absorbance at 280nm in BioKM.
11:15 – Finished 20ml elution with half of the fractions crossing the device’s upper limit (Abs>3).
11:20 – Putting twenty microliters in eppendorf tubes of selected fractions for future SDS-PAGE analysis, mixing them with 20 microlitters of Sample buffer 2x and pooled everything into a 50ml tube (except for the first elution which is contaminated from the 3rd wash leftovers). The fractions were directly diluted 1:1 in elution buffer w/o Imidazole for two reasons:
- Lowering the Imidazole concentration as this molecule can lead to protein denaturation and instability at high concentrations (need ref!).
- Lowering the protein concentration to avoid aggregation