Expression in E.coli - only 1 out of 7 constructs working! (Jun/24/2010 )
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Hi...I am back with the new results. I tried BL21 Star DE3 cells for expression. And with some modification, I could express all 5 remaining constructs! (for some reason, I left working with one). But now I have a new problem. In all these 5, my protein is in insoluble fraction! I did lot of literature searching for getting soluble protein, which forced me to do some variations in the experiments. This include, inducing below 0.5 OD, using lowest concentration of IPTG (0.1mM till 0.5mM), adding 3% ethanol just before induction, heat shock at 47C just before induction, including cofactor (Mg2+, in my case) in the media, using Novagen autoinduction media, using 2X LB-phosphate media, media with sorbitol and betaine. This, especially the last one, gave me some soluble protein, but it isn't sufficient for me, and I am seeing tons of protein in insoluble lane! Any other suggestion to convert this into the soluble form? I am somehow hesitating to denature the inclusion bodies; no guarantee of activity after refolding! Adding thioredoxin tag would be the another option, but it would require re-cloning!
how did u proceeded with ur insoluble fraction for other six clones?
I didn't get your question
Anyways...I did't do anything with the insoluble fraction of any of the 5 remaining clones. I was just trying get the protein in the soluble form. Are you trying to ask how can the protein be solubalized?
As mentioned in my previous post, out of several changes made to get the soluble protein, those done using LB media having sorbitol and betaine succeeded to some extent and right now I am proceeding with those since I get at least some protein out of it
my question is that how did u know that the protein is present in insoluble fraction? I hope u added some chaotropic agents or denaturants to the pellete fraction ( inclusion bodies),what is the exact protocol u used to check the presence of protein in inclusion bodies.
This is how I do it:
harvest 100 ml culture
re-suspend pellet in 5ml lysis buffer, sonicate
supernatant: soluble fraction
pellet: re-suspend in 500 ul lysis buffer: insoluble fraction
Use 1-2 ul of the insoluble fraction for loading on PAGE
I add regular PAGE loading buffer (which contains DTT, a protein denaturant) to all (soluble/insoluble) samples and heat them at 100C for 2 min.
The combinatorial effect of DTT and heating denatures and solubalise the protein/s in inclusion bodies
k on what basis u r deciding the amount of lysis buffer to be added for 100ml of culture.
And dont u add chaotropic agents like urea, etc to the insoluble fraction?
500ul of which lysis buffer u use to add to the insoluble fraction,
whats ur lysis buffer composition?