Re: [ccp4bb] Looking for suggestions with protein expression

[John Newitt]

On Jun 27, 2020, at 3:15 AM, Umar Farook  wrote:
> 
> 
> Dear All,
> 
> Sorry for an offtopic question, your suggestions are highly appreciated.
> 
> We have been working on iron sulfur cluster binding protein, which is usually 
> expressed as a nice soluble protein expressed in BL21 cells but aggregated in 
> the affinity column itself and unable to recover from it. We had made n 
> number of truncations and fused to soluble tags such as MBP, but always ended 
> up in large aggregates. Anyone has experience in working with iron-sulfur 
> cluster binding protein before, please let us know the critical steps in 
> purification of such proteins, whether you have completely done the 
> expression, purification and crystallization in anaerobic conditions? or else 
> changing the expression system to eukaryotic system such as Baculo or HEK 
> 293T would help?
> 
> Please share your valuable experience, thank you.
> 
> 
When you say “affinity column”, are you referring to a Ni2+-affinity column?
-John



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Re: [ccp4bb] Covalent Cysteine Aduct

[John Newitt]

There are several ways to prepare “Fabs”, using the term broadly. I would dig 
deeper in this case to be sure a thiol adduct isn’t possible.

John



> On Mar 27, 2020, at 5:38 PM, Cowan, Richard H. (Dr.)  
> wrote:
> 
> 
> Hi,
> 
> The Fab constructs have a c-terminal cysteines on both the heavy and light 
> chains, which should form a disulphide. Adding reducing agent to the 
> purification of the protein would potentially reduce this disulphide, 
> possible causing issues the stability and heterogeneity? At least that's my 
> understanding? 
> 
> Thanks,
> 
> Dr Richard Cowan
> Research Associate
>  
> HWLSB 1/05
> Department of Biochemistry
> University of Leicester
> Lancaster Road
> Leicester, LE1 9HN, U.K.
>  
> Phone +44 (0) 116 229 7077
> 
> From: CCP4 bulletin board  on behalf of Paul Emsley 
> 
> Sent: 27 March 2020 21:33
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: Re: [ccp4bb] Covalent Cysteine Aduct
>  
> 
> 
>> On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:
>> 
>> 
>>   although [BME] seems unlikely, since the crystallized protein is a Fab.
>> 
> 
> I don't follow.
> 
> 
> 
> 
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Re: [ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

[John Newitt]

Have you tried maintaining the protein in a solution with a low concentration 
of excess cofactor? Light vs dark? Excess reducing agent or different reducing 
agent or oxygen-depleted and under argon?

John

> On Feb 2, 2020, at 4:22 AM, Jon Hughes  
> wrote:
> 
> 
> Thanks for you interest. Ok, here are some more details.
> The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa) 
> holophytochrome, produced with a C-terminal His6 tag together with its 
> co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red 
> photochromic (that is, photoactive) such that, as a 2 component sensory 
> histidine autokinase / phosphotransferase, its kinase activity can be 
> switched on and off by appropriate light pulses. Thus it is unambiguously 
> functional. It is also highly soluble (10 mg/ml is no problem) – but 
> subsequently (over days and weeks) it aggregates (irrespective of the 
> photostate) to form a fluffy precipitate.
> Incidentally, I believe that most SHPK's and indeed most phytochromes have 
> aggregation problems like this.
> Beyond urea being a less potent chaotrope than guanidinium/HCl, the different 
> chemical actions of the two might give a hint as to what causes the 
> aggregation.
> Cheers
> jon
>  
> Von: CCP4 bulletin board  Im Auftrag von Artem 
> Evdokimov
> Gesendet: Sonntag, 2. Februar 2020 00:46
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] Urea vs. Guanidinium/HCl
>  
> More details would be helpful. Do you know whether your protein is folded and 
> active to begin with? Many partially folded proteins behave in a way that 
> resembles your experience... Urea is a less potent denaturant mole for mole 
> than GuHCl so it is not super surprising that it behaves differently.
>  
> Artem
>  
> On Sat, Feb 1, 2020, 6:22 PM Jon Hughes  
> wrote:
> Hello everyone,
> We work on a protein that tends to aggregate. The process is slowed but not
> stopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCl
> dissolves the aggregate readily, urea just turns it into an amorphous
> chewing-gum-like mass. Does that info provide anyone with a clue as to why
> the aggregation occurs and maybe suggest how to stop it in a way that would
> not thwart crystal formation?
> Best,
> jon 
> 
> 
> 
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Re: [ccp4bb] Off topic: room temp FPLC column cooling

[John Newitt]

Bear in mind that if you have cold buffers going into a room
temperature (or warmer) pumps, you can get outgasing that will cause
inaccurate flow or loss of priming for the pumps. You may be able to
work around by using freshly degassed eluents, but the outgasing will
still be a problem for long runs.

On Fri, Jun 7, 2019 at 11:32 AM Gianluca Cioci  wrote:
>
> Hi,
>
> a cheap solution would be to put buffers, fraction collector, and the column 
> (if not too big)
> inside a cold cabinet, next to the FPLC.
> you probably need to use longer pipes but it works
>
> Best regards,
>
> GIA
>
>
>
> Le 07/06/2019 16:51, - - a écrit :
>
> Dear all,
>
> our cold cabinet FPLC is rotting away due to the high humidity in our lab. 
> Anybody having a room temperature setup, where only the buffers, column(s) 
> and fractions are being cooled? If so, by which means (cooling spiral, water 
> pump, peltier element?). Thank you for your suggestions! Michael
>
>
> 
>
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>
> --
> 1st French Congress on Integrative Structural Biology
> Please check: http://bsi-2019.ipbs.fr
>
> Dr. Gianluca CIOCI
> BioCatalysis Team
> http://www.lisbp.fr/en/research/catalysis-and-molecular-enzymatic-engineering--ead1.html
> PICT - Plateforme Intégrée de Criblage de Toulouse
> http://cribligand.ipbs.fr/index.html
>
> Tel: +33 (0)5 61 55 97 68
> Tel: +33 (0)5 61 55 94 91
> E-mail: ci...@insa-toulouse.fr
>
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>
>
> 
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Re: [ccp4bb] Problems with an exonuclease

[John Newitt]

Probably nucleic acids. Increase the number or volume of washes and
improve the washing of your inclusion bodies. Instead of sonication,
we use a Polytron homogenizer to resuspend the IBs pellet during
washing. This is faster and easier. Incorporate an additional
chromatography step such as Heparin-Sepharose or Cation-Exchange with
SP-Sepharose if the pI of your protein supports this. Alternatively,
try to flow your protein through DEAE or Q-Sepharose in the presence
of a moderate concentration of NaCl (e.g. 200-250 mM), where much of
the nucleic acids should be captured.

- John

On Wed, Jun 7, 2017 at 9:36 AM, Mohammad Khan  wrote:
> Dear all,
>
> I am working with an exonuclease by refolding it from inclusion bodies
> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
> form.
>
> I lyse my cells using a cell disruptor and after solubilizing IBs with urea,
> I refold the protein by rapid dilution and get an aggregate and monomer peak
> of the same on GFC. and have checked CD as well as activity, both of which
> are good.
>
> My issues is as follows:
>
> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
> reach upto 2. I have tried all means to get rid of watever this
> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
> Dnase prior to lysis. I have also used methods to remove the DNA from
> protein, if that is the contaminating agent.
> I am trying to crystallize the protein with no success so far.
> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
> Orange as a fluorophore.
>
> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
> the issue of contamination and gives me good thermofluor curves. I purify
> the mutant also form IBs.
>
> Can someone suggest what this "contamination" may be?
>
> Thank you for your time.
>
>

Re: [ccp4bb] Protein-Ligand Crystallization

[John Newitt]

Hi Monica,

Your Tm results don't suggest any binding of your ligand to the protein. Your 
ligand concentrations are quite high so I am not convinced of even weak 
binding. The destabilization that you are seeing at the highest concentration 
may be due to ligand precipitation causing protein denaturation/unfolding.

If available, I would search for other ligands for co-crystallization. 
Alternatively, confirm the ligand binding by some other technique before 
investing a lot of time and protein in crystallization screening.

John

Sent from my iPad

 On Feb 5, 2015, at 8:43 AM, Monica Mittal monica.mitta...@gmail.com wrote:
 
 Hi all,
 I am working to crystallize a protein-ligand complex. I did a preliminary 
 melting curve analysis for the protein in the absence and presence of 2 
 ligands (dissolved in protein buffer). I kept the other controls as buffer an 
 a known standard to confirm instrument performance. All expts done in 
 triplicates.
 Now the results are like : Tm of protein alone is 56 deg, Tm in the presence 
 of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 
 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, 
 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !!  
 Although the effective delta Tm for both is different at higher 
 concentration, but both are kind of making protein less stable. So i was 
 wondering, will it be difficult to co-crystallize them !! Any suggestions in 
 this regard are highly appreciated !!
 
 Thanks
 Monica

Re: [ccp4bb] L-Dopa Stabilization?

[John Newitt]

No direct experience, but I think that there is some literature
supporting increased stability of L-DOPA drug formulations with
ascorbate.

- John

On Thu, Jul 3, 2014 at 1:34 PM, Keller, Jacob kell...@janelia.hhmi.org wrote:
 Dear Crystallographers,

 Does anyone have experience with co-crystallization of a protein with L-Dopa? 
 In my hands, the L-Dopa degrades in water to a dark solution within 10 
 minutes. I am thinking of trying some reductants and metal chelation, but a 
 verified successful formula would be much appreciated, and preferably it 
 would be without going into a glove box.

 Jacob Keller

 ***
 Jacob Pearson Keller, PhD
 Looger Lab/HHMI Janelia Farms Research Campus
 19700 Helix Dr, Ashburn, VA 20147
 email: kell...@janelia.hhmi.org
 ***

Re: [ccp4bb] Low 280 absorbance imidazole?

[John Newitt]

Yes, I think that's the one we use now. I believe it is the current branding of 
the Fluka imidazole that we always ordered. Definitely lower UV absorbance. We 
also prefer Roche high purity DTT for the same reason.

John

Sent from my iPad

On Aug 21, 2013, at 11:04 AM, Jan jan.abendr...@gmail.com wrote:

 Hi Bernhard,
 this one works for us:
 Sigma BioUltra imidazole, 99.5% by GC, 56749
 
 Cheers,
 Jan
 --
 Jan Abendroth
 Emerald BioStructures
 Seattle / Bainbridge Island WA, USA
 home: Jan.Abendroth_at_gmail.com
 work: JAbendroth_at_embios.com
 http://www.emeraldbiostructures.com
 
 On Aug 21, 2013, at 7:33 AM, Bernhard Rupp hofkristall...@gmail.com wrote:
 
 Hi Fellows,
  
 could someone please point me towards the source of a known high purity 
 imidazole
 with low absorbance at 280 nm? I am facing the problem of detecting a low 
 absorption protein
 in high imidazole background after IMAC gradient elution. In the UV spectra 
 of the
 2 imidazoles I checked there is some contaminant that absorbs at 280…  
  
 Thx, BR
  
 
 Bernhard Rupp
 Marie Curie Incoming International Fellow
 Innsbruck Medical University
 Schöpfstrasse 41
 A 6020 Innsbruck – Austria
 +43 (676) 571-0536
 bernhard.r...@i-med.ac.at
 
 Dept. of Forensic Crystallography
 k.-k. Hofkristallamt
 Vista, CA 92084
 001 (925) 209-7429
 b...@ruppweb.org
 b...@hofkristallamt.org
 http://www.ruppweb.org/
 ---
 

Re: [ccp4bb] Resuspension of bacterial cell pellets

[John Newitt]

I typically use a 10:1 ratio of lysis buffer to paste (e.g. 100 ml for 10 g) 
for E. coli expression and lyse by high pressure homogenization (e.g. APV at 
700-800 bar). I often add Benzonase to the lysate prior to clarification by 
sedimentation. This works great for highly expressed proteins with affinity 
capture as the first purification step.

For those who prefer to use really thick suspensions for lysis (like less than 
5:1), you should keep in mind that your pellet after clarification will contain 
a significant amount of the added lysis buffer and, consequently, the protein 
that you are trying to purify. Hence, your yield will be lower.

- John

Sent from my iPad

On Oct 25, 2012, at 8:15 AM, Raji Edayathumangalam r...@brandeis.edu wrote:

 Hello Everyone,
 
 Sorry for this rather naive and non-CCP4 question but I am very curious.
 
 My rule of thumb is to resuspend bacterial cell pellets in about 1-2% of the 
 original culture volume for a wet weight of about 3g of bacterial pellet  per 
 L of culture volume. For example, Typically, the total volume of my 
 resuspension for a 6L-bacterial cell pellet is around 60-70mL or about 40mL, 
 if I really try to minimize the volume of buffer. Every protocol I have read 
 over the years seems to indicate something similar.
 
 In troubleshooting one of my colleague's protein preps, I found that she is 
 resuspending 6L of cell pellet with a total of pellet+buffer volume of 5mL. 
 In practice, I would not physically be able to resuspend a 6L pellet in 5mL 
 (3g pellet/L culture) without making a very viscous and lumpy soup. My 
 suspicion is that such small volumes are a source of some of her issues, 
 including a high number of impurities in her elution from affinity columns.
 
 I'm curious to hear what other folks do and recommend.
 
 Cheers,
 Raji
 
 
 -- 
 Raji Edayathumangalam
 Instructor in Neurology, Harvard Medical School
 Research Associate, Brigham and Women's Hospital
 Visiting Research Scholar, Brandeis University
 
 

Re: [ccp4bb] Invisible Reference on Pubmed?

[John Newitt]

This looks like one of those journals not routinely indexed by the NLM. There 
are some issues in Medline, but not all. I only found one article from 2004.

http://www.ncbi.nlm.nih.gov/nlmcatalog/365316

John

Sent from my iPad

On Feb 7, 2012, at 4:02 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote:

 Dear CCP4BB,
 
 this is perhaps my most egregious off-topic post, but can anyone
 explain why the following reference is not findable in PubMed? I can
 get it from the ACS website, but not on PubMed or elsewhere. The
 journal is on PubMed--is it perhaps because it's funded by ExxonMobil?
 Very strange...
 
 Jacob
 
 
 Article
 A Study of the Separation Principle in Size Exclusion Chromatography
 AbstractFull Text HTMLHi-Res PDF[140 KB]PDF w/ Links[240
 KB]FiguresCiting Articles
 Thomas Sun*
 Baytown Polymers Center, ExxonMobil Chemical Company, 5200 Bayway Dr.,
 Baytown, Texas 77520-2101
 Ronald R. Chance, William W. Graessley,† and David J. Lohse
 Corporate Strategic Research, ExxonMobil Research and Engineering,
 Annandale, New Jersey 08801
 Macromolecules, 2004, 37 (11), pp 4304–4312
 DOI: 10.1021/ma030586k
 Publication Date (Web): April 29, 2004
 Copyright © 2004 American Chemical Society
 
 -- 
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***