I am expressing and purifying recombinant GFP/6His protein from Sf9 cells. Recently after what looked like a solid expression (green GFP pellet) from a 500 mL (2e6cells/mL) culture, I resuspended the pellet in 10 mL of ice cold lysis buffer and froze them as pearls in liquid nitrogen.
The 10 mL was purely empirical but I have seen protocols where it suggest 4mL lysis buffer / 1~2e7 cells. Meaning I would've had to use 200mL of lysis buffer.
I know other protocols go by pellet weight but in the end, what is the downside of having a concentrated lysate? Poor lysis? Sub-optimal purification?
Would it be recommended to dilute the frozen pellets with more lysis buffer before sonicating and purifying?
Thanks in advance
Relatively high density re-suspensions for sonication affect the sonication efficiency (lower) or other continuous type homogenizations versus single pass. There may be higher viscosity, occlusion of product, aggregation, faster protease susceptibility that in turn affect the downstream unit operations.
The problem with a too concentrated lysate is described above. The advantage is if you need to load the lysate onto a column smaller volumes are better. But with a His-tagged protein you can batch bind the protein to the resin, batch wash, and then pour the resin into a column, then the lysate volume is not an issue and that is the best way to get a protein relatively pure using a single column.
So yes, I would dilute the frozen lysate in at least 40 mL cold lysis buffer, though I use 0.05% NP-40 to gently lysis the cells (for a cytoplasmic protein), versus sonication which can denature proteins if the sample gets too warm and also breaks the nuclei open releasing lots of DNA especially if your using a baculovirus to express the protein.
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