[ccp4bb] position open to US citizens

[Zhang, Yuzhu - ARS]

https://www.usajobs.gov/GetJob/ViewDetails/576886000





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Re: [ccp4bb] Protein rapidly precipitates when off ice

[Zhang, Yuzhu]

I did not follow all the responses to this, but, did you try to use the Ni-NTA 
buffer (500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) to run the gel 
filtration?

Yuzhu.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Fage
Sent: Friday, July 14, 2017 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein rapidly precipitates when off ice

Dear All,

Thank you for the many suggestions. After sending my first message to the BB, I 
tried exchanging the sample into buffer containing 5 mM EDTA and also into 
buffer at pH 9.0 (using BICINE). Neither of these appear to have helped the 
instability--precipitation still occurred within ~1 min of removal of the tube 
from ice. The theoretical pI is ~6.1, which is far from my working pH, although 
as Mark indicated the calculated pI may be inaccurate, and I may even need to 
try a more acidic pH. I will test the ideas provided by everyone over the next 
week and leave some feedback. It may be that I need to prepare a different 
truncation, as this domain is excised from a larger covalent assembly. I have 
purified homologs trimmed at a similar position in the past, but of course that 
doesn't guarantee good behavior in my current system.

Best,
Chris

On Fri, Jul 14, 2017 at 10:20 AM, Eugene Osipov 
> wrote:
 Hi, try to add 1-5 mM EDTA, because trace amounts of metals cause 
precipitation of tagged proteins.

14 июля 2017 г. 1:40 пользователь "Chris Fage" 
> написал:

Dear CCP4BB Community,

This week, I purified a nicely overexpressing protein by Ni-NTA followed by gel 
filtration. In a 4 C centrifuge, I concentrated my gel filtration fractions to 
~1 mL, transferred the spin filter to ice, and then collected 2 uL for 
measurement on the Nanodrop. Sadly, the protein precipitated heavily in the 
pipet tip before I could dispense it onto the Nanodrop pedestal, directly 
adjacent to my ice box. This effect seems to be abated at 4 C, as the protein 
remained stable in cold room-chilled pipet tips. However, the protein also 
precipitated heavily when overnight at 4 C in 1 mL gel filtration buffer (150 
mM NaCl, 10 mM HEPES pH 7.5), but not overnight at 4 C in 10 mL Ni-NTA buffer 
(500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) prior to gel filtration. Has 
anyone experienced and resolved a similar issue before? Do any useful additives 
come to mind?

Things I have tried with the gel filtration sample:
-Exchanging buffer to restore the salt concentration to Ni-NTA levels (e.g. 500 
mM).
-Exchanging buffer to add 10% glycerol.
-Simply diluting the protein in gel filtration buffer to rule out concentration 
dependence.

In each case, the protein precipitates to a milky solution within about a 
minute of removal from ice (I am working with 20-50 uL volumes in PCR tubes).

Many thanks for any suggestions!

Best,
Chris





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Re: [ccp4bb] off topic

[Zhang, Yuzhu]

Hi Anamika,

 Proteins with high sequence identity can behave very differently when 
expressed in bacteria. Sumo (or other tags) may or may not help. If it does, 
you can express your SH2 with a tag and remove the tag after purification if 
the tag is a problem for functional studies. The vectors described in this 
article  may 
work for you if you cannot figure out why you get inconsistent expression.

YZ.


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anamika 
Singh
Sent: Thursday, July 06, 2017 7:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off topic

Hi,

Is anyone has worked with STAT1 proteins?

I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was 
no expression so far or rather say inconsistent expression. Sometimes the 
expression was in inclusion bodies.  I have tried different methods to pull out 
the protein from inclusion bodies using urea, guanidium chloride tween20 but 
none of them worked well. The yield was very low (very faint band on SDS-PAGE ) 
from 3-liter culture. I changed the host cells from BL21 to Rosetta DE3 cells 
but no success so far.

We thought to use some other vector system like with SUMO tag but did not 
proceed because the aim of the project to design inhibitor and tag will 
interfere.

Please suggest me something so that I can complete my project in lesser time.


Looking forward to valuable suggestions.

Thanks
Anamika




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