Hi everyone!
I have cloned gene in pRSETB vector and expressed in BL21(DE3) host. After fresh transformation protein is expressed. However, there are following difficulties:
1) Even only one week old glycerol stock from same colony does not express the protein.
2) Protein is expressed at 20 degrees C but not at 37 degree C.
3) Expression is not consistent.
Hi Ravindra,
BL21(DE3) are not recA endA and can therefore 'edit' your plasmid, especially so if the cells are stressed by leaky expression of a toxic protein. It doesn't happen with every plasmid and some proteins can be expressed from glycerol stocks in this strain but it is really not good practice to make glycerols in these strains. We never do it-it is too unreliable.
Keep a glycerol stock for growing plasmid in DH5 cells or similar but re-transform 'B' strains for protein expression only when you want to express the protein, keep them on plates for ca. 1 week maximum before transforming again. Addition of 1% glucose final to plates and media to really block expression until IPTG addition may also help with stability of the clone and final expression levels.
Both of these steps should improve your reproducibility.
I take it you mean that 20°C increases your levels of SOLUBLE expression-not total-and that is obviously a protein folding problem solved by te hlower temperatures as pointed out by Pierre and Yoram.
I assume you mean 2) Protein is expressed at 20 degrees C but not at 37 degree C?
If so you probably have inclusion body issues (the protein over-express and aggregate) . This is a common issue in over-expression and indeed low-temperature induction is a common solution.
Yoram
I have experienced same problem with you. I heard high glycerol concentration damages the DNA and the glycerol concentration must be lower than 10%. But I suggest you to transform the target DNA every time you need to express the protein.
I agree with Yoram about expressing at 20 °C in order to minimize the formation of inclusion bodies. You can even go lower (14 °C) if your incubator can do it. As to the inconsistent expression, your protein is probably toxic, leading to counterselection of good producers due to leakage of expression even in the absence of inducer. BL21(DE3) is notoriously prone to such leakage. Try using a host with a more stringent control of expression, such as BL21(DE3)(pLysS), or, better still, BL21AI (= arabinose-inducible) available from Thermo Fisher.
Try to make a good glycerol stock with auoclaved glycerol. Are you inducing your protein expression?
Hi Ravindra,
BL21(DE3) are not recA endA and can therefore 'edit' your plasmid, especially so if the cells are stressed by leaky expression of a toxic protein. It doesn't happen with every plasmid and some proteins can be expressed from glycerol stocks in this strain but it is really not good practice to make glycerols in these strains. We never do it-it is too unreliable.
Keep a glycerol stock for growing plasmid in DH5 cells or similar but re-transform 'B' strains for protein expression only when you want to express the protein, keep them on plates for ca. 1 week maximum before transforming again. Addition of 1% glucose final to plates and media to really block expression until IPTG addition may also help with stability of the clone and final expression levels.
Both of these steps should improve your reproducibility.
I take it you mean that 20°C increases your levels of SOLUBLE expression-not total-and that is obviously a protein folding problem solved by te hlower temperatures as pointed out by Pierre and Yoram.
How much IPTG are you using to induce? Are you using 1mM? I find that very often people use too much and driving the expression too hard results in lots of protein but most of it being insoluble.
Given that the protein appears to be soluble at lower temperatures, then reducing the concentration of IPTG may increase the ratio of soluble to insoluble protein. Try a matirx of test expressions with 0.01mM, 0.1 mM and 1 mM IPTG and at a few temperatures, 18, 20 and 25. This will help you optimise the conditions.
A squint at the specs for your vector points to Pierre's recommendation of pLysS BL21DE3 being the best strain for expression.
pRSET A, B, & C Thermo Fisher: Transformation protocol for Top10F' and BL21(DE3) introduction the pRSET vectors are pUC-derived expression. Expressed corectly and contain the N
First screen: Express a protein in BL21 (DE3) from modified pET-vectors with host strain BL21(DE3) Protein expression is induced by
I have had some limited experience with pRSET plasmids and have to say I like pET much better. I think it may be the copy number, pET plasmids may have lower copy numbers and be less likely to overload the E coli folding capacity. Also, always transform freshly for expression, like other answers suggest.
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