I have tried to express GST-p53 in E.coli (BL21-DE3, RIL, RIPL) but expression level is weak. I have come across few papers and protocols on google where researchers have purified p53 just with a His-tag.
On troubleshooting I have concluded it may be due to p53 polymorphism. Here are the details of my protein construct: NCBI-genebank BAC16799.1 (CDS@GenBank: AB082923.1). It turns out to be different from Uniprot ID: P04637-1. My protein has 72R, 273H, 309S.
Please give your suggestions.
If you have successfully purified p53 in E.coli, please share your sequence ID.
Thank you.
We may need more information for this question.
What is your method of induction? Is the protein expressed? Is the protein present in the soluble fraction following lysis? Does the protein absorb to the first resin/column, or does it flow through? Et cetera.
Confirming expression and solubilization might be a good 1st and 2nd step if the purification is failing.
My apology for writing no expression (i have edited it now). I have performed induction with 1mM IPTG at 37C and 18C. But the band intensity in SDS-PAGE is weak in comparison to the E.Coli proteins (See the gel image). I have not performed the solubility test. I will be needing good amount of protein for protein-protein interaction study and at my present expression level, I am quite skeptical to proceed further. There should be no problem with IPTG stock or physical parameter because I have got good expression for another protein which was inducted simultaneously with p53. I have also tried 2 colonies from each plate.
@Brian, Thank you for your interest. If you have any experience with p53 purification, please share your sequence ID. I want to rule out the possibility of sequence conflict of my p53 from Uniprot ID. Any other suggestions will be highly appreciated.
I have no P53 purification experience, just protein purification experience in general. While looking for expression of a band by Coomassie is a good 1st step, I have very often been able to purify large amounts of protein even when there was not a clear extra band in the induced lysate. Sometimes the induced protein is superimposed over another highly expressed protein and cannot really be seen. In such cases, I look for enrichment of a protein at the expected target mass after the 1st affinity purification step, prior to further cleanup procedures.
You could confirm expression and solubilization by Western if you want something that is more concrete than the Coomassie.
I'll leave actual purification recommendations to someone who's done a p53 purification.
Thank you for sharing your experience. I am waiting for some suggestions from community members, other wise I will proceed with small scale purification and check it out.
p53 is tough. I would recommend optimizing your time of induction, you may need to grow the cells as long as 24h post induction. Also when I have successfully expressed p53 I grew the cells at a higher temperature post induction 23 degrees C. What I would recommend is small scale optimization on time and temperature. Unfortunately, when it comes to protein expression, it really comes down to some grunt work to get the best conditions for your construct. Good luck
Thank you Sarah for your advice. I have proceeded with 1litre culture and will check the purification status. If this does not work, as you have mentioned, I need to put effort on expression conditions. Meanwhile, I was wondering if you could share your sequence ID of the p53 protein you have expressed/purified. I want to rule out sequence conflict.
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