Inclusion body Idea! Tell me what you think! - (Apr/02/2008 )

Hey,

Im working with a protein that we got made for us that provide maximal protein expression in E.coli, that is there are no codons in the protein that the E.coli cant read. Anyways i believe this is causing the BL21 cells to overexpress and form inclusion bodies. Ive tried everything from 50uM IPTG to lower temp in many combinations to get this to express, and no dice. I did however have one idea, what if i only induced with IPTG for like 15-20 min, would the cells still overexpress? Just a thought, let me know.

By the way, im trying to get the protein soluble from the start because i need to isotopically label the protein for NMR experiments. I know all about the Urea and all that, that partially works but i need the protein to be folded properly. Thanks

Andrew

Which non-optimized cells would you recommend for this?

Sorry, I assumed you meant you were using a strain like Rosetta that has rare-codon tRNA genes added. If you meant that the gene sequence itself has been optimised for E. coli, things might be more difficult. What results have you had using non-optimised coding sequence?

The current thinking is that rare codons exist in genes to allow correct folding of secondary structural elements. If you remove them all, you may adversely affect the overall folding of the protein, even destroying enzymatic activity. You cna clearly also shift production of protein into IBs.
If you do have to refold, try this site, http://refold.med.monash.edu.au/ .

HI

when u induced cell expression with IPTG and form I.B, u will also get small amount of the protein

in native and soluble form, that u can isolate and purify.

There is zero soluble protein, it all goes to inclusion bodies. I do get some when i refold it using NDSB but im not sure if its folded correctly. Being an NMR person i will make an N15 labelled protein and see if its refoled correctly. However, it currently has a His-tag on it, would replacing that with a GST-tag help solubility, ive seen that in a couple places. What about making a mutant protein construct without the optimized codons, that is making it with those codons that E.coli dont have and using the Rosetta cells then? YOu think that would work??

Andrew

How about autoinduction?

The idea is you set up your cells in media with 0.05% glucose and 0.2% lactose and when the culture gets to such a density that the glucose get used up the bacteria start to utilise the lactose instead which induces expression from your Lac promoter. This is a gentle induction which means protein is produced slowly, folds properly and doesn't end up in inclusion bodies. The method is cited in this paper:

doi:10.1016/j.pep.2006.11.022

(P.S. I've never actually used it myself but I always thought it was a really great idea!)

P

I will try that thanks alot. Anyone have any ideas about the mutation thing though or GST replacement?

GST-tagging does help the expression of insoluble proteins but it is a very large tag (about 16 kDa). What are your requirements for this protein? Will the large tag be a problem in future experiments??