Protein is in insoluble fraction during overexpression in E. coli - (Mar/07/2006 )

Hi all,

I have cloned a putative kinase from rice (Oryza sativa) in pET 28. It has an estimated size of 47 kDa including the His tag and the vector backbone. I could able to purify it in denaturing condition. To perform some bichemical assay, I do need to purify this protein in native condition. I have performed it's solubility test at different temperatures (viz. 37 oC, 32 oC and 28 oC) and four different IPTG concentration (1mM, 0.75 mM, 0.5mM, 0.25mM and 0.1 mM). In all cases, when induced cells were sonicated and the pellet and supernant were analyzed in SDS PAGE, then most of the expressed protein was found to be in pellet fraction and almost nothing is in the supernatant.


Please guide me how i would be able to make this protein soluble in E. coli.

regards,


Prasant K. Dansana

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You can start by using different expression host and maybe also to use a more inducible/control vector !

Another way would be stil to purify in denaturing conditions but then do a refolding and try to use your enzyme then ! Do you have alredy an enzymatic test to check whther your protein is functional ?

pesji

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I'm having a similar problem expressing an algal protein in E.coli, and I've tried different cell lines, and now am trying to co-express with GroEL from that organism. I've also tried refolding from Inclusion bodies, and the protein just crashes out of solution. Are there any other ideas out there?

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I try recently to express my mycobacteria protein with a pET44 vector which contains NusA a E Coli fusion partner known to be secreted into soluble fraction.

I had some protein into the soluble compartment in contrast to before where everything was in the inclusion bodies
Unfortunately production was not tremendous but you might consider this alternative 8

The vector is sold by Novagen

Pesji

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CAN U TELL ME HOW U TRY UR PROTEIN FOLDING IN INCLUSION BODY.I ALSO WANT TO DO THE SAME.
BEST RGERDS

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We routinely express proteins for structural characterization by growing the cells at 37°C until OD 600 = 0.5-0.6, adding IPTG (usually 0.3 - 1.0 mM but you will have to determine the optimal IPTG conc for your situation), and then lower the temperature to 18°C and let the cells grow overnight. Just to be safe we add 1.0% glucose to our starter cultures to try to prevent leaky expression. Alternatively, you might want to try a fusion protein. I have good luck with maltose binding protein fusions.

Good luck,
dave

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I have a similar problem with a 64 kDA fungal protein expressed in E. coli strain BL21(DE3)- we optimized induction and get a yield nice enough to do most characterisations, but not good enough to try some upscaling . Since 30-70% of my protein (depends on the variant) happen to be in the pellet (and is not refoldable because of the covalently bound FAD , we tested that), I would be glad for any advice to lower this insoluble part in favor of the soluble fraction... some tricks that don't include expensive new vectors?

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I am also trying to overexpress eukaryotic src kinase in E. coli. But all of my protein is going into the inclusion bodies. So i tried lower temp like 22 degrees and upto 0.01 mM IPTG, but that also did not help. I want these protein for doing some functional assays,, so I am not willing to go for purification with urea. Can any one please suugest me how to solubilise my protein.

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If you are having problems expressing soluble protein, you might want to check the codon bias in your construct. Rice to E. coli could be a bad jump in codon useage.

This site http://gcua.schoedl.de/
will let you see if your inclusion body problem happens because of stalled translation caused by bias.

If your problem is because of bad codon bias, go to one of the tRNA augmented strains like Rosetta (but don't take this as an exclusive endorsement, because there are plenty around).

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try expressing at 18 degrees. put your shaker in the cold room and shake at 18 overnight. it enhanced solubility in my case and other cases too. or you can refold after Urea for example. you can also precipitate with ammonium sulfate and then try to refold, i never tried it but i was told it is better than pure dialysis. Good luck!

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