On this forum I have seen questions being asked on how to reproduce expression in BL21DE3 and have found the standard advice is to freshly transform BL21DE3 cells with plasmid each time for expression. Isnt that too cumbersome? In my earlier experience we used to routinely use pET vectors glycerol stocks made in BL21DE3 and find suitable expression though I remember the proportion of protein in the supernatant and pellet would change. Please shed some light on this as I have a poorly or non expressing clone of Pet21A in BL21DE3 at 37,30 and 25 DEGREES-both uninduced and induced profiles look same.
I suppose that you designed the clone such as to fuse your polypeptide with a His tag. Thus, I suggest that you try a small-scale batch purification with a small amount of IMAC resin (e.g. 50 µl Ni-NTA pre-equilibrated with 50 mM Na-phospahte buffer , pH 8, containing 10 mM imidazole), followed by an was with the same buffer and then elution with an equal volume of 500 mM imidazole, pH 8. This will reduce the background of endogenous E. coli proteins and enable you to detect whether your protein is made at all and whether it is degraded. Don't forget to check also the insoluble fraction. As to using freshly transformed cells, I usually don' bother, but start from a glycerol stock. However, if your protein is toxic, your problem may result from leakage of expression in the absence of inducer, especially if you are using BL21(DE3) as such. Try BL21(DE3)(pLysS), or better still, BL21AI (Invitrogen), which is arabinose-inducible and affords a very stringent control.
Pierre-I have once tried purification and on lysis found the protein(expected) size in the pellet so solubilised the same and loaded on his tag and found no binding.I assumed that the clone needed sequencing and found that indeed the sequence is in frame with his tag intact so I tried re optimising expression.I am now trying with low temperature with addition of glucose to suppress basal expression.
Are you suggesting that i should still go ahead and lyse the cell pellets and load the supernatant on the column and look for binding??
Hey.. It is always very good to express protein in freshly transformed cells for protein expression ( preferably within 5 days of transformation).
U can try theses conditions for expression
induction at OD(600 nm)= 0.8-0.9, temp 37 C/IPTG-0.5 mM-1 mM/ time-3 to 6 hours
induction at OD(600 nm)= 0.8-0.9, temp 24 C/IPTG- 100 micrM-400 Micromolar/ time-12 to 24 hours
induction at OD(600 nm)= 0.8-0.9, temp-30 C/IPTG- 100 micrM-400 Micromolar/ time-8 to 12 hours
induction at OD(600 nm)= 0.8-0.9, temp 24 C/IPTG- 100 micrM-400 Micromolar/ time-12 to 24 hours.
U should lyse cells in presence of 8 M urea in buffer and run the SDS PAGE to check expression.
Possibly your protein is going to inclusion bodies which can be seen in when you lyse cells in presence of urea.
Otherwise you can express protein in BL21 codon plus strain.
All the best!
I agree with Preshant, that it would not be advisable to use a single colony that has been sitting on a plate for many days. However, I'm not sure that freshly transformed cells are necessary. I typically streak out a glycerol stock and use a single, fresh colony to start a culture.
Thanks Ronald for the answer-I am still figuring out why no expression is seen- adding glucose to the medium also didn ' t show any expressed band -I am anyway going to try fresh transformation and will try to repeat the glucose experiment and maybe low temperature to check for expression of my protein
Hi there,
Level of expression in BL21(DE3) is decreasing with ageing of the cell but also we made the observation that the level of expression may vary a lot from on clone to an other. So to avoid these problems, we systematically transform the host cell freshly, culture the mix of transformants overnight in liquid selective medium and use it for inoculating medium for expression experiment.
Thanks Dominique for the reply-on fresh transformation too I found only 2 transformants but I m going to use the same to make the O/N culture you recommended and then try for expression with and without glucose
Dominique- I tried with glucose but there is no difference between uninduced and induced lanes-please advise-we are trying to source the pLysS strain and trying with LB TB and in the presence of 1% glucose
Dominique-Im back 13 days later-with no protein both uninduced and induced look the same to me -I picked up another clone and went ahead with fresh expression studies-I read this paper in Biotechniquies on suppressing basal expression in BL21DE3
http://www.biotechniques.com/multimedia/archive/00010/00296st03_10412a.pdf
Any ideas why my protein is not showing up??
Dominqiue-I wanted to check whether there is plasmid of the recombinant in my expression strain so can I isolate the same from BL21DE3 and look for presence of insert -?Should it be done before induction or after induction?Please specify the same .
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