2 replies to this topic

[pET]

Dear all
I want to express soluble human proteins (5kD, 37kD) without any tags in pET28/BL21. I use LB, grow my cultures at 37° to OD >0.3, take a sample, then induce with 1mM IPTG and incubate for at least 3h at 30°.
This works fine in my small scale test cultures (10ml Medium in 50ml Falcon tube) - I see strong induction of my protein band with samples before IPTG / after expression on SDS Page.
It also used to work fine in large scale (700ml in 1L Erlenmeyer) some month ago.
Now I again checked small scale (same clones)- which worked fine, but could not upscale my protein expression.
In large scale I see no induction of my protein now, it even rather looks as if my protein band in SDS is stronger in the sample prior to induction (and less after induction) - it seems that there is all-over less protein in the samples after induction.
What´s the problem here ?
Please help
pET

[pDNA]

this sounds terrific!!! 700 mL in a 1 liter flask???
The problem will be that there is no head space and the aeration of the culture will be very poor ...therefore cell density will be much lower since cells will switch to anaerobic growth (have you ever compared cell densities of small and "large" scale cultivations?). Maybe your OD is lower in your large scale cultivations and therefore you see less protein? Plasmid instability under anerobic conditions could be also a problem, so it could be that  your plasmid is lost after induction in "large" scale!

I would recommend to use 250 mL in a 1 L flask and to inoculate with a overnight culture instead of starting from a single colony. You have to watch for equal biomass concentrations if you want to be able to compare your cultivations.

[Adrian K]

Hi,
Maybe I'm not helping but: Don't you think is better to express human's protein in Eukaryotic cells such as yeast rather than prokaryotic bacteria? Or maybe I'm wrong...