How did you try to detect for Lon expression? At 87 kDa there are a lot of other protein bands that it could co-migrate with making it hard to see by SDS/PAGE stained with Coomassie Blue.

Is is possible that the Lon protease is affecting E coli? If so you may need to carefully control expression (tightly regulated inducible promoter, grow e coli to medium - high density, then induction at very low temperature / aeration for many hours to days).

A control experiment is to generate a Lon expression plasmid containing a point mutation that inactivates the protease. If this expresses well then you know it's the intrinsic Lon activity that is the problem.

Good luck!

Sequence the gene in plasmid, if it is in frame, also make a western blot to check when the protein is expressing in very low quantity where you cannot observe minor expression band. you can also try different expression strains, various temperature, IPTG concentration.

Hi, expression of eukaryotic proteins can be difficult in E. coli. There are various reasons why the expression could fail. Codon usage (you used Rosetta, but you should also try Rosetta 2, or BL21 (DE3) codon plus). We very often see differences there. I guess you are using a T7 promotor driven expression system? You could try another promotor, like arabinose promotor, which can be controlles more tightly and much more fine tuned. In the case you protein is toxic for the cell you would like to keep the amount of tranacript low (use pLysS or Lemo21 cells). There are also strains like C41 or C43. If the mRNA of Lon is unstable in E.coli use Star cells. So there is a lot to consider. As Muhammad pointed out correctly, always check via immunoblot, if you overexpressed protein (soluble fraction of the cells). Good luck!

Thank you. i tried different IPTG Concentration(02,0.5,1.0 mM IPTG)at 30,37 degree temperature .I checked via immuno blot. only mutant of LONP1 is expressing but not wild type.Lon is a serine protease ,can you give some other better option/idea other than sequencing the plasmid ?what is the best way to express lagre human proteins in e.coli ?

The fact that it is a protease is the key - classic symptoms are that the non-functional mutant expresses well but the functional protease does not!. It'll be digesting E.coli proteins and making them poorly. It will be impossible to express as a functional protein in vivo at large scale. Even if you drive it to inclusion bodies you might have trouble. The growth rate of the cells carrying plasmid with functional protease will be lower than that for cells carrying mutant - have you checked this?

Some of the correspondents have suggested using the non-leaky arabinose inducible promoter - this is a good idea. Grow the cells to a high density before induction and then induce for a short time only.

I think your best bet is to try in vitro expression - but yields are low and cost is high.

Hope you are using E.coli competent cells. Try adding 1M IPTG at 37C in a shaker of 250-300 rpm for 90 mins. These should be done after you get enough O.D of the culture.

Thank you