Just to add to Herman's suggestions, if you are trying to crystallise a protease then you could also try using the S195A variant rather than an inhibitor. This would certainly be the case if you ever want to co-crystallise in a substrate, as PPACK (or the like) would occupy the active site cleft and prevent formation of the protease-substrate complex.
Tom ** > Hi John, > > This is really an amazing wild west story: the man who crystallizes faster > than his protease! I really must compliment you with how you successfully > performed these experiments! > > Unfortunately, proteins usually do not crystallize that fast (at least not > in my hands), so in these cases other methods have to be used. As has > mentioned before, protease inhibitors are the way to go. Especially with > autolysis, as one protease cuts another one, the speed of the reaction goes > with the square of the protease concentration. Whereas in dilute solutions > not much happens, as soon as you start to concentrate towards > crystallization conditions, say 10 mg/ml, degradation suddenly goes very > fast. > > There are 2 cases to consider: > 1) the protein you want to crystallize is a protease and is destroying > itself. In this case you need to cocrystallize with a potent and specific > inhibitor. With serine proteases, Wolfram Bode was very successful by using > chloromethylketone-containing peptides (e.g. PPACK). These compounds would > make covalent links with both the active site serine and histidine, > effectively killing any protease activity. > > 2) the protein you want to crystallize is not a protease and it is a > contaminant which is causing the problems. In this case I would add a > protease inhibitor coctail in an earlier step of the purification to block > the protease before the final purification steps. I would also add some > small broad protease inhibitor e.g. PMSF to the protein solution used for > crystallization. > > Herman > > ------------------------------ > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *John > Domsic > *Sent:* Wednesday, January 16, 2013 2:22 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] protein degradation in crystal > > Hi Lisa, > > Speed is definitely a big factor here. With a protein I work with I can > get large crystals in myriad conditions that only diffract to about 4-5 > Ang. What I ended up doing was taking these crystals and seeding entire > screens. I found that not only would crystals appear sooner but it > revealed novel crystallization conditions. These seeded crystals would > appear within minutes as Preben described and diffracted to better than 2 > Ang. Another thought would be to try limited proteolysis to see if you can > identify a more stable construct. > > -John > > -- > > John Domsic > Postdoctoral Fellow > Gene Expression and Regulation Program > The Wistar Institute > Philadelphia, PA 19104 > > > On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth > <j.p.mo...@ncmm.uio.no>wrote: > >> Dear Lisa >> It is not uncommon to see breakdown products when you run crystals on >> gel. Espesially if they are older crystals, sometimes you even see higher >> molecular bands, these are probably due to intra molecular cross links >> formed over time. >> If you are worried about stability, try to increase the crystallization >> speed, we have one example where we see a clear difference in both crystal >> quality and even space group depending on when we fish the crystals. The >> crystals appear within 5 min, the best quality data sets come from >> crystals we fish after only 30-60 min. >> You may also have a little protease contamination of course, to prevent >> this add protease inhibitor, or DTT, or EDTA to you protein before you set >> it up. >> cheers Preben >> >> >> On 1/16/13 12:14 PM, LISA wrote: >> >>> Hi All, >>> I have an 36KD protein which can be crystallize in two days. Most of the >>> crystals are very big. But all cystals have poor resolution,lower than 3.8 >>> A. I picked some crystals, washed them in the mother solution and then run >>> SDS-PAGE. It is surprised to find that different cystals have different >>> components. Some crystals have several samll bands below the band of the >>> protein. And in some crysals the bigger size band (as the construct should >>> be) almost disappared and have smear. Does the protein was degradated in >>> the crystals? Did someone met the similar problem as I? Thanks >>> >>> All the best >>> lisa >>> >> >> -- >> J. Preben Morth, Ph.D >> Group Leader >> Membrane Transport Group >> Nordic EMBL Partnership >> Centre for Molecular Medicine Norway (NCMM) >> University of Oslo >> P.O.Box 1137 Blindern >> 0318 Oslo, Norway >> >> Email: j.p.mo...@ncmm.uio.no >> Tel: +47 2284 0794 <%2B47%202284%200794> >> >> http://www.jpmorth.dk >> > > -- Skype: tom.murray.rust Twitter: tmurrayrust http://twitpic.com/photos/tmurrayrust +44 7970 480 601 (UK)
