Thank you all for your valuable suggestions. I will try to improve the process accordingly.
Best, Dipankar On Thu, Sep 21, 2017 at 3:18 PM, Tom Murray-Rust <tom.murray.r...@gmail.com> wrote: > Hi Dipankar, > > I've produced several serine protease constructs in Ecoli and then > refolded them. I would investigate the following (some of which echoes > previous comments): > > - try both urea and guanidine as your denaturant > - try refolding in stages (e.g. 8M urea to 2M to 100mM or equivalent) > - definitely try adding arginine to your refolding buffer (we typically > used 0.6M) > - try varying the salt concentration in your refolding buffer > - try varying your ratio of reduced:oxidised glutathione (from around 5:1 > at one extreme to 1:5 at the other) > - try refolding by diluting your denatured protein into refolding buffer > in both directions (i.e. either prepare a large volume of refolding buffer, > and have it stirring fairly vigorously, and slowly drip your denatured > protein in via a peristaltic pump over several hours; or add your denatured > protein into a large beaker with stirring, and slowly drip in your > refolding buffer over several hours) > - concentrate your refolded material by TFF over a few hours, then heat at > 37C (this will help to precipitate any protein that has refolded > incorrectly, and can improve the integrity of your final material), then > filter prior to dialysis into purification buffer > > A typical unoptimised yield would be approx 1 mg per litre E.coli culture; > with some development you may be able to get up to 10-20 mg per litre. Even > at these levels, it is not unusual to see high levels of precipitation at > various stages (refolding, concentration, heating etc). > > Also I would echo the comment about adding in inhibitors - not only can > they prevent proteolysis, but can also stabilise the active site and help > with refolding. > > Good luck with it! > > Tom > > > > > On Thu, 21 Sep 2017 at 20:14, Dipankar Manna <dipankar.biot...@gmail.com> > wrote: > >> Hi, >> >> I am working with a serine protease. As the protein is not soluble I am >> purifying it from the inclusion bodies followed by refolding. The protein >> shows good activity after refolding but the major concern is the >> 'precipitation'. Though the protein express quite well, but I loose almost >> 50-60% protein during refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, >> 100 mM KCL, 5% Glycerol, 2 mM GSH, 1 mM GSSG and 1 M NDSB) as it >> precipitates. Refolding is done in room temperature. I tried refolding at 4 >> degree, but it even end up with more precipitant. It also precipitates >> during concentrating, so in general I almost loose most of the protein >> during this refolding and concentration steps. I start with 6 lit culture >> that give around 1-2 mg protein in the final step, which I am not happy >> with. >> ​ >> Any suggestion to deal this issue would be highly appreciated. >> >> Thank you in advance. >> >> Best, >> >> Dipankar​ >> >> -- >> *Dipankar Manna, Ph.D* >> Postdoctoral Researcher >> Department of Molecular Medicine >> Institute of Basic Medical Sciences >> University of Oslo, Domus Medica >> Oslo, Norway >> >> Mob : +47 451 66 517 <451%2066%20517> >> E-mail: dipankar.ma...@medisin.uio.no <dipankar.ma...@kjemi.uio.no> >> dipankar.biot...@gmail.com >> http://www.med.uio.no/imb/english/people/aca/dipankam/ >> > -- > +61 451 402 428 <+61%20451%20402%20428> (Australia) > +44 7970 480 601 <+44%207970%20480601> (UK) > > -- *Dipankar Manna, Ph.D* Postdoctoral Researcher Department of Molecular Medicine Institute of Basic Medical Sciences University of Oslo, Domus Medica Oslo, Norway Mob : +47 451 66 517 E-mail: dipankar.ma...@medisin.uio.no <dipankar.ma...@kjemi.uio.no> dipankar.biot...@gmail.com http://www.med.uio.no/imb/english/people/aca/dipankam/
