Dear All,
I tried to express a protein in a modified M9+ (Deuterated) media as suggested in “Cai, M., Huang, Y., Yang, R., Craigie, R., & Clore, G. M. (2016). A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks. Journal of Biomolecular NMR, 66(2), 85–91. http://doi.org/10.1007/s10858-016-0052-y”.
My cells grow upto a O.D of 10 which is half of what is expected(compared to that in the article) but reasonably good enough.
However, upon lysis 60% of the protein is found in the pellet with only 40% in the soluble fraction. When expressed in H2O based media (M9) the protein is 100% in the soultion fraction.
I also find that the cells grown in deuterated M9 media somewhat harder to lyse. I had to increase the sonication amplituted to almost 80% whilst earlier I only used about 60% of the amplitude.
I have tried expressing culture at 20C and 30C @0.5mM IPTG and @0.2mM IPTG. But the IPTG and temperature do not seem to affect the fact that 60% of the protein goes into the pellet.
Does anyone seem to know how to improve the amount of protein going into soluble fraction?
some additional info : I also see white precipitate in the cell pellet (even before cell lysis) and once also during the growth of the culture. I am presuming this to be coming from some salt precipitates in the media. But I am not sure.
Thank you for your time.
One option to increase soluble protein expression is to use a cleavable solubility tag as described in the two articles below
Chapter An Evolution-Based Approach to De Novo Protein Design
Article Mocr: A novel fusion tag for enhancing solubility that is co...
You may try other hosts, like those expressing chaperones, or SoluBL21(DE3). However, you may also consider increasing the the size of your culture/biomass to be processed by 2.5-fold. I know deuterated media are costly, but so is your time.
Jeffrey R Brender Pierre Béguin Thank you for the suggestions. I will try those options if nothing at the stages of growth, expression and sonication using the current method works. FYI I am using ROSETTA-2(DE3) cells.
We are making isotopically enriched protein with 2H*13C* 15N* isotopes which makes it expensive to increase the volume of the culture and hence we are hoping to find ways of getting moreof the protein into soluble fraction.
I have found many suggestions from researchers on increasing the solubility of proteins such as the once below
They are all very valuable and very helpful suggestions. Very thank full to them.
https://www.researchgate.net/post/Can_anyone_suggest_how_I_can_make_my_protein_more_soluble
https://www.researchgate.net/post/What_else_can_I_try_to_make_my_recombinant_protein_more_soluble
https://www.researchgate.net/post/How_to_increase_solubility_of_recombinant_proteins_expressed_in_bacterial_strain
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However my Question was more in the line that
In a H2O-based media my protein is 100% soluble and the cell pellet lyses very easily. Whereas in the D2O media 70% of it goes to pellet. In addition the lysis requires more harsh conditions such as 80% amplitude of the sonicator to lyse.
So what improvements can be done where the D2O induced effects need to be minimised so as to avoid the protein to go into the pellet.
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Thank you so much for your time.
If I am reading your question correctly, the difference may be less deuteration than the switch from M9 to M9+. Does the protein express similarly in H2O based M9+?
Jeffrey R Brender I have not tried expressing the protein in M9+ H2O.
However if grown in the regular M9(D2O) the protein is in the soluble fraction. But the growth of cells was very slow (starting from a 0.05 O.D the cells reach 0.2 O.D in 10 hours) Since the cells are not growing I induce the protein at OD 0.2. which gives us roughly about 6mg of protein from 1 liter.
(please let me know if it is ok to let the cells grow for more than 24 hours and then induce? hoping that the cells will reach a OD of 0.6 in 20 to 24 hours? sometimes I also have seen the OD of the cells reducing with time which I am presuming due to cell lysis during stressful growth which is also the reason that I do not wait longer than 10 hours. )
We need proteins in the order of ~30 to 40 mg per liter for NMR studies hence we are trying media which can give good growth and soluble protein.
Thank you for taking your time.
Chandra
Jeffrey R Brender I am currently trying to add a MBP fusion tag. Meanwhile I shall also try expressing in the M9+ H2O media if it is the media that is driving the protein to the inclusion bodies. I am also trying the PG media from studier's recipies.
Thanks a lot again.
I was reading this book today and I think there are two things that you could try.
1) Double Colony Selection (to get good isotopically labeled transformed plasmids)
2) Acclimating your E.coli cells to deuterated media. (See Note 14 of Chapter 1 of the book that I attached.) Basically selecting cells at the exponential growth phase in 25% D2O media, and then transferring them to a growth plate with 50% D2O media, and then gradually increasing the D2O percentage of the growth media, monitoring the OD increase at each step.
Also, the authors say that deuterated media can have a reduced pH because of how the cells react to it releasing metabolic intermediates. Maybe that matters.
Adron Ung Ung Sorry for the late reply. I only saw your message today. Recently we had tried lowering the temperature before induction to 28C and keeping it at 28C for 15 minutes and then inducing with 1mM IPTG....at a OD of 6.0....(This is a M9plus High density optimised media whose induction point is 6.0 to 10.0 OD) This has yielded about 70% of protein in the soluble fraction. So I will keep our current protocol going.
I am thankful for your time and also for the wonderful book suggestion. Dhadchi Jeyaharan
Thank you also for your suggestion. Our protein expresses between 22C and 37C only. Next time I am planning to try 25C below that the expression levels are very low.- Badges
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