Could you identify the cleavage sites by protein sequencing and design new constructs (truncated versions) accordingly? It might improve your crystal quality to get better resolution.
Joe On Wed, Jan 16, 2013 at 9:22 AM, Tom Murray-Rust <tom.murray.r...@gmail.com>wrote: > 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) > -- Best regards, Joe
