Hi Michael, I have developed a method for stabilizing mRNA in vivo in bacteria. Normally, it is not too hard to get high levels of expression in E. coli. However, since your gene is fairly large in size it may more susceptible to mRNA degradation. Are you using protease inhibitors? See link for list of some protease mutations that could help prevent degradation in vivo.. http://www.sciencedirect.com/science/article/pii/S138917230280007X

I am using protease inhibitors after I break the cells but not during expression. 

Would you like to try a sample of my modified phosphate? What type of media are you using? Sometimes a minimal media has less protease activity and/or you could try a better protease free media.

I'm using TYP because I thought it would give high bacterial growth.  I have tried LB media in smaller growths.  I have not really compared them side-by-side though.

I would be interested in trying a sample of your modified phosphate.  

Try with other E. coli DE3 strains : BL21, BL21-Gold, C41, Origami/Shuffle, ...

Consider using other expression medium : 2xYT, SB, ...

Just to be sure I would compare the stability of your protein or a protein in minimal media vs the other medias. I can send you some material later next week. Please email the address info to [email protected].

 Hi Michael,

There is not an easy solution for your expression issues. I have some suggestions that you might try.

1. Try expressing a GST fused version of your protein. GST tags have better soluble and stable expression in E. coli than His tags.

2. Try co-expression with chaperones. Takara, Inc has/had a set of 5 plasmids with various chaperones. I have had some success with "Trigger factor" chaperone. 

3. If you happen to know another protein that binds to your unstable protein, try co-expressing them. 

Even if these work, the yield may not be much, but may be enough for some interesting biochemical work. But not enough for structural biology sort of heavy lifting.

Best

Sujay

I suggest using 20%glycerol and b-ME and high salt  from the beginning of the purification and put every ice on ice before using.

Hi,

I usually have similar expression levels and I can recover usually 1-3 mg/L after 1 step of affinity purification, which is not excellent, but usually is enough to do the work after.

I would like to focus your attention on other 3 factors: temperature, time and IPTG concentration. Are these gels from the expression at 18°C? Usually lower temperature increases much the yield. How long do you induce the expression? Time is usually very important, with low temperature you usually do an overnight expression. Did you try different IPTG concentrations?

Simone

Dr. Morra,

Thanks for the suggestions.  This gel/western was from cells expressing overnight at 18C.  I have tried expressing at 42, 37 and 18 and it appears that the yield is higher at 18C.  I have tried using different times and overnight seems to be the best.  I have not really tried varying the IPTG concentration with this construct so perhaps I will look into that.  I have a different construct that is a fusion with thioredoxin that is under the control of arabinose.  Varying the arabinose had little to no visible effect on the amount of protein I saw on a western.  The thioredoxin tag did not see to stabilize the protein either (based off of how much I saw on the western). 

Dear Michael,

it seems you already did a lot of work. If it was me, I would test temperatures such as 10, 20, 25 and 30. Sometimes in this small range there are interesting differences...

Another thing that came to my mind is: do you expect any cofactor in your protein? This usually makes a big difference.

Hi

What about the induction? how much IPTG did you used?

Did you try it with baffled expression flasks?

Hi Michael,

Could you try expression in insect cells?

You could also try auto-induction (see paper attached). It works even at 16°C.

Olivier

Hi Michael.

whit unstable proteins I always try the LEMO cells. This strain is specific for toxic proteins and the expression level is modified by L-rhamnose. You can do a expression test in small volume whit different rhamnose concentration.

for more info I attach the article !!!

Riccardo

Thanks everyone for your suggestions.  I thought I would give an update as I now have gotten a fair amount of purified protein (attached gel).  This protein came from the same strain and essentially the same media as the first gel I posted with this question except I included PLP, which is a cofactor of this protein, in the media.  I did a 4.2 L growth and I actually have a good amount of fairly pure protein after 2 columns.

I have also recently tried using a different strain (BL21-Star) and it looks to express ~2x more in small culture experiments.  I think using the cofactor and this strain will allow me to make a great deal of this protein.  

I also tried using some of Dr. Frayne's modified phosphate which she so generously provided me.  The results were intriguing but I do not think it helped increase expression in my case.  What is intriguing is the fact that the modified phosphate appeared to increase the expression of my target protein relative to the native E. coli proteins.  Unfortunately, the decreased cell growth gave me less total protein expressed.  This might be a good strategy for proteins that are difficult to purify.  Although it does not appear to be needed in my system, I encourage anyone who is having troubles with low expression to contact her about this method.