Hi everyone,
I'm currently conducting protein expression trials with a difficult to express enzyme. I'm trying a range of induction times, temperatures and induction OD. I notice that at a lower induction OD the cells seem to lyse after protein expression and even at higher ODs there's not much protein expression. There's no expression prior to induction and the cells grow fine up until this point. Is there any tips people can give me for working with toxic proteins? I've tried several cell lines including rosetta cells, c41 and standard B21 cells.
Thanks in advance
Harry
Dear Harry
is it your protein a membrane protein or a protease?
are you expressing it in the citoplasm or in the periplasm?
in case that your protein is a proteases but it is expected to be a soluble protein, if you cell lisys, that in generally correspond also to no clear surnatant when you pellet the cells after the culture, run on the gel also the media and check also if the protein is released on the media, because it is possible that a good fraction of your protein is there.
one possible solution is increase a lot the induction od, for example by use a media suitable for high cell density as the Enpresso B media and perform induction at low temperature (17C)
you can found a details infornation about enpresso b media and protocol on my blog https://proteocool.blogspot.com/?m=1
ProteoCool n °5 : E. coli protein expression using high cell density media
ProteoCool n°7: High cell density protein expression using Enpresso B
in case that those strategies do not work you can or perform mutagenesys in other to deactivate the catalitic site of your protease/toxic or leave e.coli for an expression host as brevibacillus, pichia pastoris or expi 293 mammalian cells which are able to secrete the protein outside the cells and theoretically are less subjected to their activity.
if your protein is a membrane protein you have to find a way to slow down ora expression rate. if c43 or temperature are not enough, you can try to use a pladmid based on a weaker or more modulable promoter than t7 as rhamnose or arabinose.
good luck
Manuele
Hi Harry, I don't know what enzymatic activity your protein of interest has, but I would consider targeting the enzyme to there periplasm in order to avoid the negative effects that could produce in the cytoplasm.
Check this paper: Article Efficient production of active chicken avidin using a bacter...
Good luck :)
There might be two different problems here. One is toxicity, and you seem to be handling it fine since the cells do not lyse when uninduced and you observe no problems when transforming the construct. The other is low expression levels, and it is not necessarily related to toxicity. Have you tried checking the translational initiation efficiency of the construct in something like the RBS calculator? Also, you say you checked Rosetta strains; does the gene have an unusual codon composition? If it does, take into account that sometimes rare tRNA supplementation does not solve the problem and it is better to resort to a synthetic, codon-adjusted gene.
Hope it helps!
I agree with Alejandro Martin, if your target protein is toxic, how does the colony grow during the transformation? You can try to optimize the codon. In addition, please be sure to check your lab environment and reagents for phage contamination, which can also affect bacterial growth and protein expression.
All the best!
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