Re: [ccp4bb] Sv: [ccp4bb] Co crystalization with less soluble ligand.

[Amit Meir]

Some ligands/ small molecules solubility is pH- dependent. (E.g. biotin, EDTA)
Try adjusting the pH (it doesn't have to be accurate, just use a pH
paper if you have very small volumes). It might help you out to
improve ligand solubility (and binding with your protein).
Also, not to ruin the party, but have you done a base line ITC
measurement (protein to buffer, no ligand) and substracted it from the
protein - ligand measurement? It might improve your calculated kd
slightly, or, alas, you'll realise the measured affinity was due to
protein's denaturing upon interaction with the solvent.

Good luck,
Amit


Quoting Jon Cooper <488a26d62010-dmarc-requ...@jiscmail.ac.uk>:


I realised afterwards that this might be an experimental example of
desperately trying to turn a negative result into a positive one,
which is something that has been discussed here recently (sorry,
Dale) ;-0 ;-0

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 Original Message 
On 24 Apr 2021, 14:33, Jon Cooper wrote:


If you want to try co-crystallisation again, if you dissolve your
ligand in say DMSO or anything that works, e.g. isopropanol, then
add it to your protein up to its maximum tolerable level (i.e. the
level up to which the protein is not denatured/inactivated by the
solvent - you need to test this), the ligand will start to
precipitate out on a grand scale but don't worry, stir the mix at
4°C for a couple of days so that the ligand can slowly partition
into the protein binding sites, hopefully. Then you can dialyse out
the organic solvent and/or use one of the spin concentrators to
remove it and concentrate the protein for crystallisation. There
will be tons of precipitated ligand which you can centrifuge out
whenever necessary.

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 Original Message 
On 24 Apr 2021, 13:08, abhimanyu singh wrote:


You may soak the crystals with ligands in maximum tolerable DMSO
percentage and harvest crystals at different time points. In my
experience working with some poor affinity, highly hydrophobic
compounds, it may take up-to several days for the ligand to bind
with good occupancy (in one instance it was four days). This may
also depend on the crystal soaking/growth temperature.
Ethylene glycol is a good alternative to DMSO in some cases, which
might particularly be helpful in co-crystallisation experiments.

Greetings,
Abhi

On Sat, 24 Apr 2021 at 09:11, Casper Wilkens
<5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk> wrote:


How long did you wait before freezing the crystal? Sometimes I
have to wait days before the ligand finds it way into the binding
site.

Casper Wilkens
Asst. Prof.
Structural Enzymology & Biorefining
DTU Bioengineering

Technical University of Denmark

Department of Biotechnology and Biomedicine
Søltofts Plads
Building 224
Room 028
2800 Kgs. Lyngby
[c...@dtu.dk](mailto:c...@bio.dtu.dk)
www.bioengineering.dtu.dk/
https://www.cazypedia.org/index.php/User:Casper_Wilkens
https://twitter.com/protein_artist
https://www.researchgate.net/profile/Casper_Wilkens
https://www.cazypedia.org/index.php/User:Casper_Wilkens

---

Fra: CCP4 bulletin board  på vegne af
Gourab Basu Choudhury 
Sendt: 24. april 2021 07:40:20
Til: CCP4BB@JISCMAIL.AC.UK
Emne: Re: [ccp4bb] Co crystalization with less soluble ligand.

I tried sokaing ,high concentration of DMSO is effecting crystal,
so I tried other contidtion where 10%DMSO is there. Got 1.9 A
data with protein co crystalization with 10mM ligand. But ligand
occupancy is not visble.
I got the KD value from ITC, with reverse titration. With 10uM
ligand in cell and 150uM protein in syringe. .

Can anybody suggest some views.please feel free to share your experiences.

On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing,
Parkville)  wrote:


Hello Gourab,

DMSO is not the only possible solvent- there are others to try.
I don't want to sound negative, but 40 micromolar is not a very
tight binding compound. It would also depend on how that binding
constant was measured- how did someone get enough in solution to
measure that?
Best of luck, tom

Tom Peat, PhD
Proteins Group
Biomedical Program, CSIRO
[343 Royal
Parade](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g)
[Parkville, VIC,
3052](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g)
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

---

From: CCP4 bulletin board  on behalf of
Gourab Basu Choudhury 
Sent: Saturday, April 24, 2021 12:46 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Co crystalization with less soluble ligand.

Hello everyone,

I am finding it difficult for getting a ligand density inside
the protein as the ligand is very much insoluble. It's only
soluble in 100% DMSO. I tried for co crystalization. Kd value of
the ligand is near 40um. Any suggestion what to do?

Re: [ccp4bb] Dear All

[Amit Meir]

Dear Amala,
1. Increase your buffer pH. As a rule of thumb, usually it is 
recommended to work with pH 7.6 and above for Ni purification. Try pH 8.
2. Increase your buffer A [NaCl] to 500mM. Some proteins are more stable 
in higher NaCl, and for many, 50mM is not enough. It is possible your 
protein has already precipitated in the lower [NaCl]. You can also 
increase glycerol to 10%.


Having said that, you didn't provide additional details (Protein's 
origin - eukaryotic/ prokaryotic) - if it's from a eukaryotic source, it 
could be your protein needs additional post translation modifications 
(e.g. sugars etc) for stabilization, which most likely will require a 
different expression system. If your protein is of a bacterial source, I 
would try to add a solubility tag (eg MBP), change the location of the 
His tag, or change expression conditions (lower temp, different strain).

There are plenty of other options, but I'd start with that.

Good luck,
Amit



On 03/01/2018, 05:56, amala mathimaran wrote:


I am trying to purify a protein using HIS-select Ni-affinity column 
(washing with 50mM Tris pH7.5, 50mM NaCl, 30mM imidazole, 3mM BME, 
5%glycerol and elution with the same buffer + 250mM imidazole, 500mM 
Nacl). The protein (pI= 6.04) becomes cloudy/precipitated within 
15minutes of after elution. I add EDTA to the eluted protein 
immediately in the fraction tubes but the protein still precipitate, 
can anyone suggest how to avoid precipitation of protein.



Thanks and Regards,




--
*
Dr. Amit Meir
Professor Gabriel Waksman's group
Institute of Structural and Molecular Biology
Department of Biological Sciences
Birkbeck College
Malet Street
London WC1E 7HX
UK

Re: [ccp4bb] Inclusion Bodies

[Amit Meir]

Hi,
Well, sometimes proteins don't express even though they are from the same 
family and their "cousin" behaved very well. 
I found the followings helpful:
1. forget about the concept "this special strain *should* work because it is a 
Rosseta/ pLysS etc." Sometimes it's a long mRNA, sometimes a rare codon, 
sometimes it's an additional disulfide bond. If you are happy with the 
inclusion bodies protocol (and you know your protein is active later), just 
scan additional strains that you have in the lab (I like the BL21 DE3 BLR, 
which are specialized for long mRNA, but I found them useful in "regular" genes 
as well).
2. Scan "high" temperature (37C) in different time scales - maybe your cells 
need overnight expression instead of 3/4/5hrs? Or maybe the protein undergoes 
degradation overnight, so it is better to grow it for 3hrs only?
3. Change the medium to 2XYT or TB, and increase IPTG concentration if you work 
with a pET vector.
4. Try inducing in OD=1 instead of 0.6.
5. Add glucose (0.1%, but you can optimize) to the media to repress leakiness 
of a toxic protein.
6. Look for "autoinduced media" protocol. The additives there can make a 
difference between none to a few milligrams/liter.
7. Some mutations occurred in the expression vector. Re-clone to a fresh 
plasmid (same/ another pET vector/ vector with a different inducer, e.g. 
arabinose).
8. From my experience, sometimes E.coli get along with non-optimized eukaryotic 
genes even better than the optimized sequence. I would go to additional 
optimization as one of the last options.
9. A mammalian homolog? Or try expressing in lower temperature (17/20/23C) to 
get a soluble protein? (I know sometimes this is not an option).


A starting protocol would be several different strains (everything you and your 
neighbors have in the labs, basically), in 2XYT + 0.1% glucose, induction in 
0.5-1mM IPTG, then grow 3hrs/5hrs/overnight in 37C. Alternatively, 
2XYT+autoinduced media + 0.1-0.2mM IPTG when OD=0.6 to "boost" expression even 
further (and different times as well). 

Good luck :)

Amit




Dr. Amit Meir
Institute of Structural and Molecular Biology
Department of Biological Sciences
Birkbeck College
Malet Street
London WC1E 7HX
UK