Hi, One more suggestion would be autoinduction. This may not help in all the conditions but still can be checked. Instead of IPTG use 0.2% lactose in media and check for expression at different temperature. Hope it helps. All the best. On 17 Oct 2014 21:55, "Roger Rowlett" <[email protected]> wrote:
> You've tried the obvious things. Lowering the IPTG concentration doesn't > really work all that well, at least in my hands. We find we can still get > maximal expression from many promoters like trc with only 0.2 mM IPTG, and > the response doesn't modulate well: it's either on or off. Growing at lower > temperature, like 18-22 deg C sometimes works, but sometimes not. Here are > a few more approaches: > > 1. Use "leaky" expression in a pET vector system. Just don't induce it > at all and grow for 4-18 hours. The slow expression may allow for protein > folding before kinetic trapping in protein tangles. You may want to combine > this with lower temperatures and less rich media (e.g., LB instead of TB) > to slow growth and protein synthesis. > 2. Put multiple, tandem copies of your gene behind the promoter. I'm > not sure how this works, but it may tie up enzymes transcribing the message > from the plasmid to slow mRNA transcription and thus translation. I never > really understood the chemistry behind this, and no one could explain it to > me in a cohereht way. However it works, it can slow protein expression > enough to allow for soluble, instead of precipitated protein. This works > like a charm to express the alpha subunit of trp synthase. The vector has 4 > tandem copies of the gene after a trc promoter. You can make 1000 mg of > soluble protein per 100 mL of culture (!) (I didn't mess up my zeros.) > 3. Express your protein with a solubilizing tag on the N-terminus. I > think there are some commercial vectors that put NusA in front of your > gene. This may allow for soluble expression. NusA is supposedly better than > 6X His tagging for making soluble protein. (Hasn't worked for me yet, but > the literature suggests some successes.) > 4. Try a different promoter. We use a modified version of pTrc99 that > has at trc promoter instead of the T7 promoter. It is tightly controlled by > IPTG, with no leaky expression. It can be a fierce promoter, but it may not > be as fierce as T7, and can give you different results. > > Have fun! And good luck! > > _______________________________________ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: [email protected] > On 10/17/2014 8:51 AM, xaravich ivan wrote: > > Dear cc4bb enthusiasts, > This is slightly off topic but many protein crystallographers might be > familiar with this problem. > > I have been trying to over-express a bacterial (non-E.Coli) protein in > E.Coli and more than 80% goest to inclusion bodies. > > I tried the following > > Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 > mM) > > Cold shock for 30 minutes in ice before induction > > Slow rotation speed at 18 degrees O/N after induction > > While these steps helped a bit I still get about 50-60% of my protein in > inclusion bodies. > > I would like to know what other steps do you suggest to enhance the > yield in the soluble fraction (without changing the host strain or > manipulating the DNA) > > Thanks in advance > Ivan > > >
