Hi there,

Is there no expression at all or expression as insoluble protein?

Hi,

Have you tried expressing at 18 C for 18 hours? And 1 mM IPTG should be plenty

@dominique The first induction I sonicated and the protein wasnt expressed at all. I'm not sure if the standard conditions apply to a protein of that size.

@Simanga After so many hours...doesn't the protein become toxic??

Hi Diego

Usually using the standard conditions I did express proteins like 14kDa, 18 kDa or 50 kDa using bacteria BL21(DE3) in LB medium at 30C with 0,5mM IPTG (4 hrs) or  at 20 C, Overnight. When you suspect a protein to be expressed is toxic the bacteria usually degrade it, If this happens your culture must be grown in the presence of glicose then exchange the medium with LB medium + 0,5 mM IPTG, this is important because LB medium contain lactose, then  it start the expression before you add IPTG.  Another proposal is to test another strains.

An important suggestion is verify if the DNA fragment cloned into the vector is in frame with the histidine tail sequence

Hi Diego!

I recommend you to run a western blot with the standard conditions first. Because sometimes the expression might be very less than you expect. 

Hi Diego,

I have expressed 13KDa proteins at 37 degree Celsius,adding 1mM IPTG when OD at 600nm is 0.8 , and grown for five hours. But the thing is I work with membrane proteins which go into the inclusion body. So that's where the similarity ends. However I also have worked with soluble proteins, albeit they were slightly larger. You could try expressing your protein at a lower temperature for longer durations. Like at 18 degree Celsius overnight, or 25 degree Celsius for seven or eight hours. Also you can try different IPTG concentrations like 0.1 mM, 0.2mM, 0.5 mM, 1mM and 1.2mM.Optimize and proceed.

And is your protein toxic in nature? In that case try expressing in BL21 (DE3) pLysS strain. This will help prevent leaky expression prior to induction. If you want to go ahead with BL21 (DE3) then add glucose (5g/L) to the media to reduce/ prevent leaky expression.

I don't think small size of protein (15kDa) will be much of an issue. Keep trying and if you have problems please contact.

Best of Luck.

Hi Diego,

There are lots of other variables you can play with to try and optimise your expression. Things worth checking: 

- your cell line. Are you using BL21? Perhaps you could try another e.g. Rosetta. Are there any characteristics that would make your protein's expression difficult? Is it a membrane protein, have lots of disulfide bonds, or other PTMs? There may be strains that can deal with these better.

- your growth medium. You could try rich growth media (TB, 2xYT) versus a comparatively poor medium like LB or even Minimal. My own protein expresses better in minimal growth medium and I get no expression in TB. If you're particularly concerned about the induction you can also try auto induction media. 

- the transformant. Are you using freshly transformed cells? Transformants should ideally be only 1-2 days old when you inoculate your overnights, otherwise they don't work so well. Glycerol stocks can also misbehave and eject their plasmids etc. I'd also check to make sure your expression construct is what you think it is if you haven't already. 

Good luck,

James

Low your induction temperature like induce protein at 16C, 18C or 22C temp. because at low temperature the small size protein folding occur more accurately as compare to 37C. I have experience to express small size protein expression. Also reduce the amount of IPTG like 0.1-0.2 mM 

I hope must work.