[ccp4bb] off topic - UV absorbance of phosphotyrosine
Hello folks, I would like to measure the concentrations of proteins containing phosphotyrosines using absorbance at 280nm. I'm wondering about calculating extinction coefficients by the Edelhoch method - as used in ProtParam for example: http://web.expasy.org/protparam/protparam-doc.html Given that phosphotyrosine absorbs less strongly at 280nm than tyrosine, has anyone developed a modified extinction coefficient calculation that treats tyrosine and phosphotyrosine separately and has the same kind of accuracy that Edelhof achieved using amino acid analogues (e.g. comparing glycyl-L-tyrosylglycine to the phophorylated equivalent)? Cheers, Will.
Re: [ccp4bb] KD of dimerization, off topic
Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fitting for the rest of us: http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes - and presumably concentrations if the KD is small enough - are feasible in an ITC200. The protein is recoverable anyway. All the best, Will. On 14 February 2014 12:40, Williams, John Charles jcwilli...@coh.org wrote: Sedimentation equilibrium or sedimentation velocity experiments by analytical centrifugation is the best method for this. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS Nicolas [nicolas.f...@synchrotron-soleil.fr] Sent: Friday, February 14, 2014 12:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina - *SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p) -
Re: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex
Hello Anindito, While Nick and Paul have thrown you some great ideas around the TAP-tag theme (although GST may not be ideal as it dimerises itself), I feel that I've read your initial e-mail differently from them. Maybe you could clarify? It sounds like you have a specific tag of ~5kDa in mind, presumably for a specific purpose? It's not the case that any old tag will suffice? Are you producing the dimeric protein recombinantly? If so, you have lots of options. If not, you're going to need a chemical modification post-purification (or to establish a recombinant system). Does the insertion of the tag need to be at a specific site within the one protein? Would one of the termini do? Critically, is your dimer a homodimer? If it's a heterodimer, things could be quite easy. All the best, Will. On 27 August 2013 18:50, Paul Paukstelis shocksofmig...@gmail.com wrote: Nicholas is right that it depends a lot on the dimer you are working with. I worked on a dimeric tRNA synthetase and was able to make heterodimers in a couple of ways. The most effective was to make a bicistronic version of the ORFs each with their own S-D. Each had a tag for tandem affinity purification as Nick suggests. In my case, using two plasmids or even putting the two ORFs with separate promoters did not result in heterodimers (probably due to folding/dimerization during translation?). I was able to express to the two dimeric versions separately, mix, dilute extensively (which ultimately resulted in a big hit in yield), concentrate, and purify through the tandem approach. I eventually noticed that even though it was slow, there was dimer exchange occurring at intermediate concentrations. I ended up engineering a disulfide at the dimer interface to minimize that. --paul On 08/27/2013 01:10 PM, Noinaj, Nicholas (NIH/NIDDK) [F] wrote: Anindito, First off, I have never tried this, but here are a few initial thoughts on how I might approach this. But first a question, how tight is the dimer form? if you mixed dimers with tags and dimers without tags, would you get any exchange? If so, you might be able to use this to your advantage. But for now, i'll ignore this. Now for how i was thinking of possibly accomplishing the goal you want... 1- use two vectors or a DUET vector to express two versions of your protein, (1) with N-term cleavable (TEV site?) HIS tag and your fusion protein, and (2) with N-term cleavable (TEV site?) GST tag only. 2- express your protein as normal and then purify by (1) running the GSH column to capture all GST tagged proteins, followed by a (2) Ni-NTA column to capture those with HIS tags. If all works as planned, you would end up with one monomer having only HIS-fusion protein tags and one having GST tag. You could use SDS-PAGE/Western blot analysis to verify this, since the monomers will run at different sizes (you might have to modify your gel system to get the best separationi recommend 5-7% gels for this size in MES) AND should be reactive to antiHIS and antiGST antibodies in needed. Further, you could get an idea for the ratios of each by comparing the overall intensity of the bands under normal staining methods. 3- to remove the HIS and GST tags, just treat with TEV protease to yield your dimer with only one of the monomers containing your fusion protein. I am sure i likely overlooked something in my haste, but you get the idea, basically a two step purification with two tags. Again, I haven't tried this but seems like it should work to me. hope this helps, good luck! Cheers, Nick --**-- [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.**gov/buchanan/http://www-mslmb.niddk.nih.gov/buchanan/ __**__ From: Anindito Sen [andysen.to...@gmail.com] Sent: Monday, August 26, 2013 11:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex Dear All, I need to insert a tag protein of ~5 kDa in one of the molecules of a dimer protein (size is ~100kDa). To be more precise - The tag need to get itself attached only on one of the dimer molecules. My expertise are not in Cell biology and therefore any suggestion in this regard will be of great help. Thanks Best Wishes Dr. Anindito Sen (Ph.D) Structural Biology Graduate School of Medicine University of Tokyo 7-3-1 Hongo. Bunkyo-ku. Tokyo 113-0033. Japan Tel Fax: +81-3-5841-3339
Re: [ccp4bb] Expression of large proteins in E. coli
Hi Nick, A couple of other suggestions: (1) Try an E. coli K strain (e.g. JM109). They're different in glucose metabolism (JN Phue et al, 2005, Biotechnol Bioeng 90:805820) and in my experience tend to grow more slowly - which seems to help improve yields of large constructs. (2) Optimal oxygenation also seems to be very important in producing large constructs. Not sure what vessels you're using but Fernback flasks work well for me. Or a fermenter might solve all you problems. Good luck, Will. Hello All, I apologize for the non-CCP4-related query. I have been working for several weeks now trying, with limited success, to express some very large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli. Limited success means I have expressed enough soluble protein to see on a gel, but not enough to purify. I have tried the obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the process of subcloning into vectors for (1) SUMO fusion and (2) periplasmic expression (pET26b). I get the sense from digging through the literature that high level expression of large proteins depends mostly on the individual protein and I will ultimately have to look for homologs. But, this is my first experience expressing such large proteins and I am curious to know if anyone out there has some magical trick they wouldn't mind sharing. Thanks in advance, Nick - Nicholas R. Silvaggi, Ph.D. University of Wisconsin-Milwaukee Department of Chemistry and Biochemistry 3210 North Cramer Street Milwaukee, WI 53211 Phone: 414-229-2647 Email: silva...@uwm.edu
[ccp4bb] Research Technician/Associate
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