Dear Sheila

your question is complex because it depends a lot from the intrinsic properties of your protein ( eg isoelettric point, presence of cysteines or hydrophobic regions) and from the domain that you clone.

of course reduction in the induction temperature as you done can improve in some cases protein solubility and if with 6h at 20C you obtained some improvements i think you can try to extend the induction time up to 16h and decrease the temperature at 17C.

if you are using lb media, generally i'm growing thd bacteria at 37C up to OD about 0.6 afterwards i shift the temperature at 17C and i induce with iptg O/N.

if the trial in lb at 17C will give you nice results in terms of solubility but low yiels as mg/l, you can try the enpresso media which will allow to you to reaxh very high cell density also at 17°C and therefore scale down the culture volumes.

you can found more information about Enpresdo on my blog: ProteoCool https://proteocool.blogspot.com

if the test at 17 fail you have to consider to:

- try different lysis buffer

- use a fusion tag as mbp the can enhance solubility

- re-design the protein domain or insert mutation in case it is an enzime and the in-solubility can be due to enzime toxicity for e.coli cells.

however if you already saw done improvements from the test at 20° C i'm oprimist.

good luck

Manuele

You could try decreasing the concentration of IPTG. Less is more because bacteria can't metabolize IPTG. You could also try decreasing the temperature still. I set my shaker to 16°C which actually is corresponds to 12°C when measured with an alcohol thermometer and sometimes I get good expression. Perhaps try overnight expression at low temperature because cell growth for E.coli is slower at lower temperatures so it should seem that it would take longer to get higher yield.

Hi Sheila,

Looks like changing the temperature and also increasing the incubation time helps. So to add on what Manuelle and Adron have just answered -

1) if you were not using baffled flasks then you may want to consider using them as it provides aeration

2) Use the appropriate E.coli strain if you have S-S bonds in you proteins..For S-S bonds formation you can use Origami cells among others.

3) Try to even lower the temperature using strains with cold-adapted chaperones to ultimately obtain more folded proteins in the soluble fraction

4) Try different media formulations, including 2X YT or even autoinduction medium.

Perhaps, trying mini expression trials (10 mL culture) at different condition followed by optimization will not be a bad idea before going back to the drawing board to redesign your construct.