I am working with a GPCR and intend to mass express the N-terminal which is like 224aas for immunological assays. Initially we cloned it in pET15b and put a 6xHis tag at the C-terminal (27kDa) but we were not able to get a expression since we always ended up purifying a contaminant. We have switched to pET28a+ with the N-terminal tag (31.6 kDa) and we have optimized the codons to improve expression in BL21 DE3 pLysS but still not getting anything. What can I do?
Hi Robert,
First question : you say that you haven't successfully purified the protein, but have you observed any actual induction using any method? By this I mean induction that is visible on typical Coomassie-stained SDS-PAGE rather than something sensitive like Western blots so that there is significant protein to work with. I am assuming from what you've written that the sequence is the extracellular N-terminal 224aa domain not containing transmembrane segments.
Related to this - which expression methods have you used? Medium? Temperature? IPTG []? Auto-induction? Additives?
Second question - from what species (or uniprot ID) does your sequence originate? You say that you have optimised codons for E.coli , but using which algorithm or method?
Have you considered trialling a modified BL21 such as C41 DE3 or C43 DE3? I use these regularly, and although the growth rate is usually a bit slower than BL21 DE3 under the same circumstances, they can often express things that make BL21 (and derivatives such as pLysS) not very happy.
Please come back and discuss these questions further!
bonne chance!
Hello Dickinson, thanks for your response. Throwing more light like you requested: I used autoinduction for my expression after I could not see any difference with IPTG-induced and non-induced cultures. The results talked about here are from Coomasie Blue staining. The 224 amino acids are just for the ectodomain excluding any TM and the protein is from Onchocerca. Codon optimisation was done using an algorithm from GenScript as they synthesized the gene for us.
At the moment, I am considering trying with Rosetta-gami 2 DE 3 pLysS, as I don't have C41 or C43 DE3. Actually thought of working with NiCo21 DE3 since it eliminates most of the the contiminant we have had in previous attempts. I am also doing WBs with anti-His Abs and Abs against a short peptide within the portion we are expressing.
When you mention the contaminant - one protein or protein class that is quite common when expression is sub-optimal is chaperone - Hsp70, GroEL, DnaK etc that are expressed as part of the unfolded protein response. These can cause real problems because they co-purify with your protein, sometimes in stoicheometric excess. Sometimes this arises when expression is actually too effective and the protein is translated so rapidly (because of the codon optimisation) that it does not fold correctly.
I have used Rosettagami(2) DE3 on many occasions, particularly good for human sequences although many others express. It is my second choice of alternative away from classical BL21(DE3) instead of C41(DE3) - traditionally I use all three of these strains in parallel when optimising.
I was unaware of NiCo21 - thanks for describing this.
Another thought : which resin are you planning to use when you have induction? I have had a lot of recent success using Cobalt- NTA resin as the background binding is very low (even in the absence of Imidazole) compared to Ni- resin ; however, you need to be aware that your protein can elute at lower concentrations of Imidazole than would be typical for the equivalent Ni- resin, so adjust your programme / fraction collections accordingly until fully optimised.
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