[ccp4bb] off-topic: short oligo concentrator
Hi all, We're planning to use short oligos to co-crystallize with protein. Basically we will synthesis that from company and then purified by ion-exchange then concentrate it. Does any one know a easy way or product to concentrating short oligo (such as 5nt)? The only way came to my mind, is to desalt it by dialysis first, then use SpeedVac. I will appreciate for any suggestion. Best, Xiao
Re: [ccp4bb] A quick question about making PEG cryo-protectant
Hi Mayer and Jason, Thanks for your quick reply! I read from Hampton that for PEG smaller than 5K, directly increasing its concentration is a way of making cryo. But you're right, glycerol or PEG400 will be a good way and might be easier. I'll try that first. Thanks a lot! Best, Xiao 2014-10-13 17:59 GMT-07:00 Mayer, Mark (NIH/NICHD) [E] : > Try either glycerol or PEG 400. > Increasing concentration of 'high' MW PEG is not a good strategy. > > ____ > From: Xiao Xiao [victor41...@gmail.com] > Sent: Monday, October 13, 2014 8:51 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] A quick question about making PEG cryo-protectant > > Hi, > > Since my crystallization condition contains PEG, I am trying to use PEG as > one of my cryo for testing. For example, one crystallization condition is > 0.2M Na2PO4, 20%PEG3350. I want to make 0.2M Na2PO4 and 40% PEG3350 as > cryoprotectant. When I added 200ul salt from 1M stock, and 800ul PEG from > 50% stock, mixed them, seems they stayed as emulsion (confirmed by > microscope), no matter how I pipette or vortex them. I tried to incubate it > at 40degree, seems it got worse. > > I am now trying to re-make the cryo from solid reagent, not solution > stock. Will this be helpful? Or is there any other trick? > > I will really appreciate! > > Best, > Xiao >
[ccp4bb] A quick question about making PEG cryo-protectant
Hi, Since my crystallization condition contains PEG, I am trying to use PEG as one of my cryo for testing. For example, one crystallization condition is 0.2M Na2PO4, 20%PEG3350. I want to make 0.2M Na2PO4 and 40% PEG3350 as cryoprotectant. When I added 200ul salt from 1M stock, and 800ul PEG from 50% stock, mixed them, seems they stayed as emulsion (confirmed by microscope), no matter how I pipette or vortex them. I tried to incubate it at 40degree, seems it got worse. I am now trying to re-make the cryo from solid reagent, not solution stock. Will this be helpful? Or is there any other trick? I will really appreciate! Best, Xiao
Re: [ccp4bb] off-topic;Crystal cannot dissolved in buffer
Hi, Thank you all for replying and giving me great suggestion in such a short time! This is a great community. Here I try to summarize the replies I got, just in case if someone is interested: 1. Of course, the most straightforward and reliable way is to test it on the beam, period. 2. Since beam may not be available every time, another simple way is to use glass filer/metal needle/cat whiskers to poke crystal. Protein crystals usually easy to shatter, salt crystals are hard like rocks. And crosslinked crystal (especially happens when crystal is not fresh, or PEGs solution is old) is rubber-like, easy to bend but hard to break. Crosslinked will reduce resolution of diffraction. 3. Can tell whether it is protein or salt by IsIt (0.1% methylene blue) staining, glutaraldehyde crosslinking/staining (causing protein crystals to become yellow due to Schiff base formation). 4.Some people did mention, in a few cases, some protein crystals were hard to dissolve. 5. Using low pH(3-5), NaOH, or simply add SDS loading buffer and heat may help dissolve the crystal. Again, thanks a lot for all your help! Best regards, Xiao Xiao 2014-09-25 16:53 GMT-07:00 Xiao Xiao : > Hi everyone, > > Sorry for an off-topic question. > > I got a problem with crystal dissolving. Basically I got crystals of my > protein in various conditions, most conditions contain PEGs but different > salts. These crystals has very similar shape, so it should not be salt. > > Now I am trying to dissolve the crystal to make sure it is my protein, by > SDS-PAGE and N-term sequencing. I washed the crystals in its original > crystallization buffer few times then transfered them into regular buffer > (500mM NaCl, 50mM HEPES 7.5) with or without 10mM DTT, however the crystal > didn't dissolve. > > I then tried to heat it then add SDS loading buffer to run a gel, I did > see very small amount of protein on the gel, at the correct position, but > it's not enough for N-term sequencing. > > Is it normal for a protein crystal? And does anyone have any suggestion > for dissolving such crystals? > >
[ccp4bb] off-topic;Crystal cannot dissolved in buffer
Hi everyone, Sorry for an off-topic question. I got a problem with crystal dissolving. Basically I got crystals of my protein in various conditions, most conditions contain PEGs but different salts. These crystals has very similar shape, so it should not be salt. Now I am trying to dissolve the crystal to make sure it is my protein, by SDS-PAGE and N-term sequencing. I washed the crystals in its original crystallization buffer few times then transfered them into regular buffer (500mM NaCl, 50mM HEPES 7.5) with or without 10mM DTT, however the crystal didn't dissolve. I then tried to heat it then add SDS loading buffer to run a gel, I did see very small amount of protein on the gel, at the correct position, but it's not enough for N-term sequencing. Is it normal for a protein crystal? And does anyone have any suggestion for dissolving such crystals?
Re: [ccp4bb] Pipettes for initial crystallization screening
Thank you all for your reply! Here I summarize what I got from the answers: 1. Seems single channel pipette is a better choice than multi-channel with small volumn. 2. Positive displacement pipette is more advantageous than regular air displacement pipette. 3. Tips with repellent surface is also helpful. FYI, following are what I got recommended: Gilson microman (positive displacement, but the minimum volume is 1ul) http://www.gilson.com/Pipette/Products/44.224/Default.aspx#.U5Yqx5SSyG8 Hamilton syringe in a PB-600 clicker (need to work with Gastight syringe together) http://www.hamiltoncompany.com/products/syringes/c/211/ PICUS pipette (both single and multi channel has small dispensable volume <1ul) http://www.biohit.com/us/liquid-handling/pipettes-electronic/products/70/picus-electronic-pipette Eppendorf Repeater pipette OR just a regular Eppendorf 2.5ul pipette seems ok http://www.sigmaaldrich.com/catalog/product/aldrich/z683779?lang=en®ion= Rainin 20 uL single channel electronic repeating pipette (Need to refill every 20ul, but can dispense 0.5ul consistently). All of them are great, I think currently a repeating pipette or Hamilton syringe is just good enough for my purpose. I'll discuss with my colleague to decide which model we are gonna purchase. Again thank you all for your suggestion! Best, Xiao 2014-06-09 14:41 GMT-07:00 Xiao Xiao : > Dear all, > > Here's a very basic question -- > I am doing some initial crystallization screening now but unfortunately > our robot got broken and it will take a while to get funded for buying a > new one:( So I have to set up all trays by hand. While it is still durable > to use our current pipettes sets, which are usually fine to dispense > 0.7ul-1ul per drop (not very accurate I think), I am wondering do you have > any recommended pipettes with smaller volume that meet my requirements? > > We are using Axygen 96-well sitting-drop plates for initially screening. > For the mother liquor I usually use regular multichannel pipette and it > works well, so I am seeking the one for the drop: > > 1. Multi-channel pipette that can accurately dispense about 0.7-1ul drop. > 2. Single pipette that can accurately dispense smaller drop size, such as > 0.4ul to 0.8ul. > > Thank you! > Best, > Xiao > > >
[ccp4bb] Pipettes for initial crystallization screening
Dear all, Here's a very basic question -- I am doing some initial crystallization screening now but unfortunately our robot got broken and it will take a while to get funded for buying a new one:( So I have to set up all trays by hand. While it is still durable to use our current pipettes sets, which are usually fine to dispense 0.7ul-1ul per drop (not very accurate I think), I am wondering do you have any recommended pipettes with smaller volume that meet my requirements? We are using Axygen 96-well sitting-drop plates for initially screening. For the mother liquor I usually use regular multichannel pipette and it works well, so I am seeking the one for the drop: 1. Multi-channel pipette that can accurately dispense about 0.7-1ul drop. 2. Single pipette that can accurately dispense smaller drop size, such as 0.4ul to 0.8ul. Thank you! Best, Xiao
Re: [ccp4bb] Oligomerization of maltose-binding protein
Hi Zhijie, Thank you so much for the reply. The protein I am working on is from eukaryotic cytosol. We've already tried optimize the IPTG concentration and induction temp. Co-expression of chaperons seems to be a interesting idea, I googled and found this one: http://www.clontech.com/takara/US/Products/Protein_Research/Protein_Folding_and_Expression/Chaperone_Plasmid_Set?sitex=10031:22372:US Do u think it is good? Or do u have other recommendations? Thank you very much! Best, Xiao On 2013-11-14, at 下午5:14, Zhijie Li wrote: > Hi Xiao, > > The tail sequence does not look very hydrophobic. But until your experiments > is done we can’t really say for sure that is not the cause for the > aggregation. > > If your protein has cys residues then if the protein molecules were misfolded > and were forming intermolecular disulfide links, adding 1-10mM DTT without > denaturation may not be able to resolve the disulfide linked multimers, > because the disulfide could be buried in the misfolded proteins. You can run > SDS PAGE with samples boiled with or without DTT to see if you have > intermolecular disulfide links. > > Since your protein contains cys residues, I wonder what is the source of it. > Is it eukaryotic secreted protein or ER/Golgi protein? If the protein is > eukarytic cytosolic/nuclear protein you do not need to worry about the > oxidative folding of them, and producing in the cytosol of E. coli might be > fine. But if the protein was eukaryotic secreted protein that contain > disulfide bonds, then you have to find an expression system that have the > proper chaperone systems to let the peptide form correct disulfide linkages. > In such case, yeast, insect, or even mammalian expression system should be > tried. > > The linker between your protein and MBP is important. You do need long enough > linker for making sure the two domains are not interfering each other’s > folding. This requirement is protein-specific though. The poly Asn linker > provided by the pMal vectors is normally long enough. According to your > description that your protein family members were not even soluble with other > tags, I think it is quite clear that these proteins are not well behaving in > your current expression system. So unless you detect serious problems in your > construct design, I do not recommend you to invest too much effort on trouble > shooting the linker and so on because this is likely not the main problem > here. > > If you have to make it in e.coli, expression condition has to be optimized to > maximize their chance to fold into soluble proteins. These optimization may > include: 1) slow down the expression by using less IPTG or using lower temp; > 2) to co-expression chaperones(there are many reported cases this works like > magic); 3) to co-express the target protein’s binding partners/cofactors; 4) > to use special expression strains such as the NEB shuffle strain that allows > cytosolic oxidative folding of disulfide-containing proteins, or the rare > codon-expressing strains to resolve rare codon problems if any; 5) to send > your protein to periplasmic space (use pMAL-p vectors instead of pMAL-c) if > you believe the protein should contain disulfides > > Or you can try to refold the protein in vitro. In such case you’d better use > the non-MBP tags, and get the inclusion bodies as the start point. > > But after all, I always prefer to use expression systems that’s most close to > the protein’s natural habitat. There are proteins that would never be folded > in E coli. > > Zhijie > > > > From: Xiao Xiao > Sent: Thursday, November 14, 2013 2:18 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Oligomerization of maltose-binding protein > > Thank you for the replies! > > Rana: Thank you for your prompt reply! I have the same problem, based on my > SDS-gel, I also have that 42kD MBP band when purifying my fusion protein. > Fortunately I can separate that from my protein of interest by size-exclusion > column. So the major issue I have now is the oligomerization of my fusion > protein. And it is also weird that I got the free-MBP as a large oligomer. > > Zhijie: Thank you for your very detailed and well-written suggestions. It is > very interesting that the MBP fusion protein can form 'micelles'. We found > very similar properties when cloning other family members into MBP vector, > they all have a very large oligomer peak, around 10-mer (several hundreds > kD), but not in void volumn of sup6 column. Biologically these proteins could > form oligomer but not this big. We have tried to purify these protein in > other tags, but most of them were insoluble. Only MBP tag gives us such a
Re: [ccp4bb] Oligomerization of maltose-binding protein
Thank you for the replies! Rana: Thank you for your prompt reply! I have the same problem, based on my SDS-gel, I also have that 42kD MBP band when purifying my fusion protein. Fortunately I can separate that from my protein of interest by size-exclusion column. So the major issue I have now is the oligomerization of my fusion protein. And it is also weird that I got the free-MBP as a large oligomer. Zhijie: Thank you for your very detailed and well-written suggestions. It is very interesting that the MBP fusion protein can form 'micelles'. We found very similar properties when cloning other family members into MBP vector, they all have a very large oligomer peak, around 10-mer (several hundreds kD), but not in void volumn of sup6 column. Biologically these proteins could form oligomer but not this big. We have tried to purify these protein in other tags, but most of them were insoluble. Only MBP tag gives us such an impressive solubility. But I agree -- they may be still misfolded. As I replied to Rana, I agree with you that the oligomerized free-MBP is really strange. That's why I want to figure out the reason, to exclude the possibility that the tag itself causes aggregation. Reading-frame should be correct since SDS-gel indicates the peptide length is correct. I didn't put the stop codon right after precision site, so there are some restriction cutting sites got translated. The exact C-term should be 27aa (LEVLFQGP+HMSMGGRDIVDGSEFPAGN). Do you think this could be the reason that MBP get oligomerized? I am trying to delete these C-term tail to see how it will behave. And the answer for your last question: there is one Cys supposed to be reduced on the surface of my protein (others are internal and really conserved), but I include 1mM DTT during purification and have tried even 10mM DTT when running gel-filtration but it didn't get improved. I have one more question. Let's say if the aggregation is really caused by the property of this construct, could it be possible to help its folding by modifying linker region between MBP tag and my protein? I know it could be very tough, but is there any general idea or strategy that we can try as a start point, such as change hydrophobicity? Again, thank you all and I am looking for your responds! Best regards, Xiao 2013/11/13 Zhijie Li > Hi Xiao, > > MBP usually is monomeric, unless you put something really nasty to its > ends. People have mentioned before that due to the high solubility of MBP, > MBP tag can drag otherwise insoluble/overly hydrophobic protein domains > into solution. Then this half hydrophilic half hydrophobic molecule can > form micelle-like structures. > > > "the small peaks are both free-MBP monomer, but the big peaks are fusion > protein and free-MBP respectively." > > So your fusion protein is never found in the monomer peak? That strongly > hints aggregation caused by the domain you fused to MBP. The free MBP > monomer peak you see on the MBP-fusion run is likely a proteolytic product > of the fusion protein, or is the natural MBP from E. coli (although > normally far less than the over expressed MBP fusion in quantity). Are you > certain that your target protein domain is not naturally oligomerizing? If > it is not or only dimerizes or trimerizes, you might need to consider > modifying the construct or expression strategy (including moving away from > bacteria if necessary). I would suggest against trying to rescue an > inherently unhappy construct or trying to make a protein in a system that > does not fold it. > > The high MW peak for the "free" MBP is a little strange. I produced > MBP-TEV cleavage site(ENLYFQG) fusion as my control before, never observed > any non-monomer species of it. the precision site does not seem any worse > than the TEV site by sequence. (But did you put a stop codon right after > the precision site? What is the exact sequence of your C-term tail? Also > double check your plasmid sequence for frame-shift mutations, since you > have modified the vector.) Forming heterogeneous aggregates of 10+ the > monomer size is indicative of misfolding if it is not caused by a > hydrophobic tail. For robust proteins such as MBP, such bad aggregation > suggests that something in your expression or purification procedure needs > to be optimized. Inducing for too long (eg, >3hr at 37C) or harsh lysis can > contribute to misfolding. > > One more question: does your protein contain cys residues? Are they > supposed to be oxidized or reduced? > > Zhijie > > > > -Original Message- From: Xiao Xiao > Sent: Wednesday, November 13, 2013 5:20 PM > To: CCP4BB@JISCMAIL.AC.UK > > Subject: [ccp4bb] Oligomerization of maltose-binding protein > > Dear all, > > I am purifying a fusion protein with maltose-bindin
[ccp4bb] Oligomerization of maltose-binding protein
Dear all, I am purifying a fusion protein with maltose-binding protein(MBP) as the N-term tag. As a control, I also purified free-MBP from empty vector. The vector I used is pMAL-c5x (from NEB) with an addition of precision protease cutting site (8 residues, LEVLFQGP) between MBP and multi-cloning site. Then I run both protein through Superose6(size-exclusion chromatography) to check oligomerization states. To my surprise, both protein has two peaks, one is at the monomer size and another one has a big MW. SDS-gel shows from both protein, the small peaks are both free-MBP monomer, but the big peaks are fusion protein and free-MBP respectively. The estimated MW of the big peaks are similar to 10 folds of their corresponding monomers but heterogeneous (checked by DLS and native gel). To my knowledge, MBP should behave as a monomer. Does anyone have seen the similar thing before or could give some explanations? I suspect maybe the precision protease cutting site and the following multi-clonging site at the C-term of free-MBP might cause oligomerization, could it be possible? I am trying to delete this part to see whether it will improve, is there any other thing I can try in order to eliminate heterogeneity? Any suggestion or comment will be really appreciated. Thank you! Best regards, Xiao Xiao
