I am expressing transcription factors in E.coli. Sometimes after the lysis (Lysosome and ultrasound) I see transparent gel like matter in my lysate which can't be cleared by centrifugation (18 000 g for 15 min). It could be genomic DNA? Every time I see this my yield is very low. Can someone explain why this can happen and how to avoid it?
Dear Max,
I suppose that you verified that your protein(s) is expressed or overexpressed by running a small volume of the culture by SDS-PAGE? Besides, the yield of a cloned transcriptional factor depends on its solubility. Check the pellet fraction on the presence of your protein. There are several ways to increase the yield poorly soluble (aggregated) proteins.
However, it is quite possible that the transcription factor of your interest is linked to a genomic DNA (and RNA). Two simple ways to override the problem.
1. Add DNase (i.e. low concentration of bensonase) to degrade DNA before centrifugation if you do not need the purified protein in further DNA binding assays.
2. Use 2-3 times "freezing-defreezing" approach by 15-30-min storing the lysed culture at -80°C then at 4°C to degrade high molecular mass chromosomal DNA (before centrifugation). It helps also to eliminate traces of RNA.
Both approaches could provide the better yield of your protein (if tagged) by affinity purification.
Dear Vehary,
thanks for the suggestions! However, I need my transcription factors for DNA-binding assays (after purification, in vitro). Why would you think that I could no longer use them if I added a DNase?
Max,
Traces of DNase will cleave a DNA probe at the extremities if you use PCR product or at the nicks introduced if you use circular DNA. It means that an additional step is required to eliminate DNase.
In your case, if you use the affinity purification method, freezing-defreezing of the culture before centrifugation appears to be the simplest way to improve the yield of your protein. Attention, transcriptional factors usually provide the DNA binding ability/specificity as dimers or other oligomeric forms. Check your protein migration in native PAGE to confirm its functionality before bi ding assays.
Best wishes
I used homogenizers like this before in addition to sonicator for lysis of E.coli:
https://www.fishersci.co.uk/shop/products/borosilicate-glass-homogenizers/p-8010615
Genomic DNA is destroyed mechanically when moving the pestle up and down. The sample becomes less viscous. Only disadvantage: you need a lot of muscle. Maybe somebody in your building has the electric version.
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