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 <[email protected]> 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: [email protected] <[email protected]> > [email protected] > http://www.med.uio.no/imb/english/people/aca/dipankam/ > -- +61 451 402 428 (Australia) +44 7970 480 601 (UK)
