Re: [ccp4bb] Precipitation issue during refolding

[Dipankar Manna]

Thank you all for your valuable suggestions. I will try to improve the
process accordingly.

Best,

Dipankar

On Thu, Sep 21, 2017 at 3:18 PM, Tom Murray-Rust 
wrote:

> Hi Dipankar,
>
> I've produced several serine protease constructs in Ecoli and then
> refolded them. I would investigate the following (some of which echoes
> previous comments):
>
> - try both urea and guanidine as your denaturant
> - try refolding in stages (e.g. 8M urea to 2M to 100mM or equivalent)
> - definitely try adding arginine to your refolding buffer (we typically
> used 0.6M)
> - try varying the salt concentration in your refolding buffer
> - try varying your ratio of reduced:oxidised glutathione (from around 5:1
> at one extreme to 1:5 at the other)
> - try refolding by diluting your denatured protein into refolding buffer
> in both directions (i.e. either prepare a large volume of refolding buffer,
> and have it stirring fairly vigorously, and slowly drip your denatured
> protein in via a peristaltic pump over several hours; or add your denatured
> protein into a large beaker with stirring, and slowly drip in your
> refolding buffer over several hours)
> - concentrate your refolded material by TFF over a few hours, then heat at
> 37C (this will help to precipitate any protein that has refolded
> incorrectly, and can improve the integrity of your final material), then
> filter prior to dialysis into purification buffer
>
> A typical unoptimised yield would be approx 1 mg per litre E.coli culture;
> with some development you may be able to get up to 10-20 mg per litre. Even
> at these levels, it is not unusual to see high levels of precipitation at
> various stages (refolding, concentration, heating etc).
>
> Also I would echo the comment about adding in inhibitors - not only can
> they prevent proteolysis, but can also stabilise the active site and help
> with refolding.
>
> Good luck with it!
>
> Tom
>
>
>
>
> On Thu, 21 Sep 2017 at 20:14, Dipankar Manna 
> wrote:
>
>> Hi,
>>
>> I am working with a serine protease. As the protein is not soluble I am
>> purifying it from the inclusion bodies followed by refolding. The protein
>> shows good activity after refolding but the major concern is the
>> 'precipitation'. Though the protein express quite well, but I loose almost
>> 50-60% protein during refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2,
>> 100 mM KCL, 5% Glycerol, 2 mM GSH, 1 mM GSSG and 1 M NDSB) as it
>> precipitates. Refolding is done in room temperature. I tried refolding at 4
>> degree, but it even end up with more precipitant. It also precipitates
>> during concentrating, so in general I almost loose most of the protein
>> during this refolding and concentration steps. I start with 6 lit culture
>> that give around 1-2 mg protein in the final step, which I am not happy
>> with.
>> ​
>> Any suggestion to deal this issue would be highly appreciated.
>>
>> Thank you in advance.
>>
>> Best,
>>
>> Dipankar​
>>
>> --
>> *Dipankar Manna, Ph.D*
>> Postdoctoral Researcher
>> Department of Molecular Medicine
>> Institute of Basic Medical Sciences
>> University of Oslo, Domus Medica
>> Oslo, Norway
>>
>> Mob   : +47 451 66 517 <451%2066%20517>
>> E-mail: dipankar.ma...@medisin.uio.no 
>>dipankar.biot...@gmail.com
>> http://www.med.uio.no/imb/english/people/aca/dipankam/
>>
> --
> +61 451 402 428 <+61%20451%20402%20428> (Australia)
> +44 7970 480 601 <+44%207970%20480601> (UK)
>
>


-- 
*Dipankar Manna, Ph.D*
Postdoctoral Researcher
Department of Molecular Medicine
Institute of Basic Medical Sciences
University of Oslo, Domus Medica
Oslo, Norway

Mob   : +47 451 66 517
E-mail: dipankar.ma...@medisin.uio.no 
   dipankar.biot...@gmail.com
http://www.med.uio.no/imb/english/people/aca/dipankam/

Re: [ccp4bb] Precipitation issue during refolding

[Tom Murray-Rust]

Hi Dipankar,

I've produced several serine protease constructs in Ecoli and then refolded
them. I would investigate the following (some of which echoes previous
comments):

- try both urea and guanidine as your denaturant
- try refolding in stages (e.g. 8M urea to 2M to 100mM or equivalent)
- definitely try adding arginine to your refolding buffer (we typically
used 0.6M)
- try varying the salt concentration in your refolding buffer
- try varying your ratio of reduced:oxidised glutathione (from around 5:1
at one extreme to 1:5 at the other)
- try refolding by diluting your denatured protein into refolding buffer in
both directions (i.e. either prepare a large volume of refolding buffer,
and have it stirring fairly vigorously, and slowly drip your denatured
protein in via a peristaltic pump over several hours; or add your denatured
protein into a large beaker with stirring, and slowly drip in your
refolding buffer over several hours)
- concentrate your refolded material by TFF over a few hours, then heat at
37C (this will help to precipitate any protein that has refolded
incorrectly, and can improve the integrity of your final material), then
filter prior to dialysis into purification buffer

A typical unoptimised yield would be approx 1 mg per litre E.coli culture;
with some development you may be able to get up to 10-20 mg per litre. Even
at these levels, it is not unusual to see high levels of precipitation at
various stages (refolding, concentration, heating etc).

Also I would echo the comment about adding in inhibitors - not only can
they prevent proteolysis, but can also stabilise the active site and help
with refolding.

Good luck with it!

Tom




On Thu, 21 Sep 2017 at 20:14, Dipankar Manna 
wrote:

> Hi,
>
> I am working with a serine protease. As the protein is not soluble I am
> purifying it from the inclusion bodies followed by refolding. The protein
> shows good activity after refolding but the major concern is the
> 'precipitation'. Though the protein express quite well, but I loose almost
> 50-60% protein during refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2,
> 100 mM KCL, 5% Glycerol, 2 mM GSH, 1 mM GSSG and 1 M NDSB) as it
> precipitates. Refolding is done in room temperature. I tried refolding at 4
> degree, but it even end up with more precipitant. It also precipitates
> during concentrating, so in general I almost loose most of the protein
> during this refolding and concentration steps. I start with 6 lit culture
> that give around 1-2 mg protein in the final step, which I am not happy
> with.
> ​
> Any suggestion to deal this issue would be highly appreciated.
>
> Thank you in advance.
>
> Best,
>
> Dipankar​
>
> --
> *Dipankar Manna, Ph.D*
> Postdoctoral Researcher
> Department of Molecular Medicine
> Institute of Basic Medical Sciences
> University of Oslo, Domus Medica
> Oslo, Norway
>
> Mob   : +47 451 66 517 <451%2066%20517>
> E-mail: dipankar.ma...@medisin.uio.no 
>dipankar.biot...@gmail.com
> http://www.med.uio.no/imb/english/people/aca/dipankam/
>
-- 
+61 451 402 428 (Australia)
+44 7970 480 601 (UK)

Re: [ccp4bb] Precipitation issue during refolding

[Debasish Kumar Ghosh]

Dear Dipankar, 

Your problem is quite tricky to solve. I have two opinions which worked for me 
for a very high aggregation prone protein (Huntingtin, which always used to go 
in inclusion bodies and precipitate even in elution buffer). 
1. First have 4-5M Guanidinum Hydrochloride in the lysis buffer during the 
purification process, and keep 150mM proline in the elution buffer. I have 
experienced that addition of proline greatly reduces the aggregation 
propensity. 
2. Second option is dealing the problem with advantage of that problem. Try to 
precipitate the protein (which is in elution buffer) with acetone (soon after 
elution) and resolubilize it in your desired final buffer very rapidly under 
cooling conditions (like in 4 degree centigrade). Though the 100% protein may 
not resolubilize, but it will fairly solubilize as maximum as up to 90% (as 
that happened with me). My intuition is that in the final solubilized fraction 
you will get high proportion of folded form of your protein. 
Just check if it works for you. 

Best wishes!! 

Debasish 

CSIR- Senior Research Fellow 
C/o: Dr. Akash Ranjan 
Computational and Functional Genomics Group 
Centre for DNA Fingerprinting and Diagnostics 
Hyderabad, INDIA 

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com 
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) 
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html 



- Original Message -

From: "Dipankar Manna"  
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Thursday, September 21, 2017 3:42:28 PM 
Subject: [ccp4bb] Precipitation issue during refolding 

Hi, 

I am working with a serine protease. As the protein is not soluble I am 
purifying it from the inclusion bodies followed by refolding. The protein shows 
good activity after refolding but the major concern is the 'precipitation'. 
Though the protein express quite well, but I loose almost 50-60% protein during 
refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 
mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room 
temperature. I tried refolding at 4 degree, but it even end up with more 
precipitant. It also precipitates during concentrating, so in general I almost 
loose most of the protein during this refolding and concentration steps. I 
start with 6 lit culture that give around 1-2 mg protein in the final step, 
which I am not happy with. 
​ 
Any suggestion to deal this issue would be highly appreciated. 

Thank you in advance. 

Best, 

Dipankar​ 

-- 
Dipankar Manna, Ph.D 
Postdoctoral Researcher 
Department of Molecular Medicine 
Institute of Basic Medical Sciences 
University of Oslo, Domus Medica 
Oslo, Norway 

Mob : +47 451 66 517 
E-mail: dipankar.ma...@medisin.uio.no 
dipankar.biot...@gmail.com 
http://www.med.uio.no/imb/english/people/aca/dipankam/ 

Re: [ccp4bb] Precipitation issue during refolding

[Jared Sampson]

Hi Dipankar - 

I will echo Thuy's suggestion of higher glycerol concentration in your 
refolding buffer.  A protocol I used in a previous lab (for refolding antibody 
ScFv fragments) steps down gradually over 3 days from 10%-5%-0% glycerol in the 
dialysis buffer, from an initial sample of inclusion bodies dissolved in 6 M 
GdnHCl and diluted to 2.4 M GdnHCl with Tris buffer.  Your protein may need 
even higher concentrations.

Killikelly A, Zhang H-T, Spurrier B, et al. Thermodynamic Signatures of the 
Antigen Binding Site of mAb 447–52D Targeting the Third Variable Region of 
HIV-1 gp120. Biochemistry. 2013;52(36):6249-6257. doi:10.1021/bi400645e.

Hope that helps,

Cheers,
Jared

> On Sep 21, 2017, at 6:38 AM, Thuy Ngo 
> <1612d752f0ee-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Dear Manna,
> 
> I can see that you use only 5% glycerol in your buffer. This concentration is 
> quite low for non-stable protein. I would suggest you to increase glycerol up 
> to 20%. I had a case before that I need to use 30% glycerol in order to 
> decrease the precipitation. Also, how did you do the refolding? 
> 
> Best luck

[ccp4bb] AW: [ccp4bb] Precipitation issue during refolding

[Herman . Schreuder]

Hi Dipankar,

Are you expressing the active protease, or the inactive precursor (zymogen)? 
Although we never systematically looked at it, I strongly suspect that many 
active proteases destroy the expression host and you will therefor only find 
clones expressing the protein in inclusion bodies.

Upon refolding, autoproteolysis may become a problem. Since it is a bimolecular 
process, the rate goes up with the square of the concentration. So during the 
concentration steps, you may have to add a strong inhibitor of your protease. 
If it is a new structure, you could consider adding an irreversible, covalent 
inhibitor to completely inhibit your protease. PPACK has been used for this in 
a large number of proteases, but it depends on the substrate specificity of 
your protease. Alternative, you could mutate the active site serine into an 
alanine.

So in summary:
-produce the protease as a zymogen and activate afterwards. There is a reason 
nature produces most proteases as zymogens.
-make sure you prevent autolysis during refolding and especially during 
concentration.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dipankar 
Manna
Gesendet: Donnerstag, 21. September 2017 12:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Precipitation issue during refolding

Hi,

I am working with a serine protease. As the protein is not soluble I am 
purifying it from the inclusion bodies followed by refolding. The protein shows 
good activity after refolding but the major concern is the 'precipitation'. 
Though the protein express quite well, but I loose almost 50-60% protein during 
refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 
mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room 
temperature. I tried refolding at 4 degree, but it even end up with more 
precipitant. It also precipitates during concentrating, so in general I almost 
loose most of the protein during this refolding and concentration steps. I 
start with 6 lit culture that give around 1-2 mg protein in the final step, 
which I am not happy with.
​
Any suggestion to deal this issue would be highly appreciated.

Thank you in advance.

Best,

Dipankar​

--
Dipankar Manna, Ph.D
Postdoctoral Researcher
Department of Molecular Medicine
Institute of Basic Medical Sciences
University of Oslo, Domus Medica
Oslo, Norway

Mob   : +47 451 66 517
E-mail: dipankar.ma...@medisin.uio.no
   dipankar.biot...@gmail.com
http://www.med.uio.no/imb/english/people/aca/dipankam/

Re: [ccp4bb] Precipitation issue during refolding

[Thuy Ngo]

Dear Manna,

I can see that you use only 5% glycerol in your buffer. This concentration is 
quite low for non-stable protein. I would suggest you to increase glycerol up 
to 20%. I had a case before that I need to use 30% glycerol in order to 
decrease the precipitation. Also, how did you do the refolding? 

Best luck

Re: [ccp4bb] Precipitation issue during refolding

[Corbin Black]

Hey Dipankar,

There are various additives for improving solubility. I would consider some of 
these:

- denaturants (ie urea, guanidine)
- aggregation suppressors (arginine, low concentrations of urea, etc)
- folding enhancers (sucrose, ammonium sulfate)

And have you tried refolding your protein on the column, during purification? 
This has the advantage of allowing you to transition into any number of buffer 
systems as you go from the unfolded to folded state, without having 
protein-protein interactions that may otherwise cause precipitation.

Corbin Black, BSc
MSc Biochemistry Candidate
University of Northern British Columbia
Department of Chemistry
bla...@unbc.ca
blackcor...@gmail.com
250-552-5046

Sent from my iPhone

On Sep 21, 2017, at 3:14 AM, Dipankar Manna 
> wrote:

Hi,

I am working with a serine protease. As the protein is not soluble I am 
purifying it from the inclusion bodies followed by refolding. The protein shows 
good activity after refolding but the major concern is the 'precipitation'. 
Though the protein express quite well, but I loose almost 50-60% protein during 
refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 
mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room 
temperature. I tried refolding at 4 degree, but it even end up with more 
precipitant. It also precipitates during concentrating, so in general I almost 
loose most of the protein during this refolding and concentration steps. I 
start with 6 lit culture that give around 1-2 mg protein in the final step, 
which I am not happy with.
?
Any suggestion to deal this issue would be highly appreciated.

Thank you in advance.

Best,

Dipankar?

--
Dipankar Manna, Ph.D
Postdoctoral Researcher
Department of Molecular Medicine
Institute of Basic Medical Sciences
University of Oslo, Domus Medica
Oslo, Norway

Mob   : +47 451 66 517
E-mail: dipankar.ma...@medisin.uio.no
   dipankar.biot...@gmail.com
http://www.med.uio.no/imb/english/people/aca/dipankam/

[ccp4bb] Precipitation issue during refolding

[Dipankar Manna]

Hi,

I am working with a serine protease. As the protein is not soluble I am
purifying it from the inclusion bodies followed by refolding. The protein
shows good activity after refolding but the major concern is the
'precipitation'. Though the protein express quite well, but I loose almost
50-60% protein during refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2,
100 mM KCL, 5% Glycerol, 2 mM GSH, 1 mM GSSG and 1 M NDSB) as it
precipitates. Refolding is done in room temperature. I tried refolding at 4
degree, but it even end up with more precipitant. It also precipitates
during concentrating, so in general I almost loose most of the protein
during this refolding and concentration steps. I start with 6 lit culture
that give around 1-2 mg protein in the final step, which I am not happy
with.
​
Any suggestion to deal this issue would be highly appreciated.

Thank you in advance.

Best,

Dipankar​

-- 
*Dipankar Manna, Ph.D*
Postdoctoral Researcher
Department of Molecular Medicine
Institute of Basic Medical Sciences
University of Oslo, Domus Medica
Oslo, Norway

Mob   : +47 451 66 517 <451%2066%20517>
E-mail: dipankar.ma...@medisin.uio.no 
   dipankar.biot...@gmail.com
http://www.med.uio.no/imb/english/people/aca/dipankam/

[ccp4bb] Postdoc position at the FHS Macau - William Chao Lab

[williamchao]

The Faculty of Health Sciences (FHS) at the University of Macau is seeking a 
highly motivated Postdoctoral Research Fellow to perform structural studies on 
key cell-cycle protein complexes.

The position will be part of the laboratory of William Chao, who focuses on 
using structural biology and recombinant systems to investigate crucial 
cell-cycle mechanisms. The laboratory has extensive experience in determining 
multi-subunit protein complexes and nurtures a collaborative spirit in pursuing 
important biological questions.

The appointee will be given the opportunity to master state-of-the-art 
techniques in protein complex reconstitution and structure determination as 
well as performing experiments at domestic and overseas collaborators’ 
laboratories.

For further information, please contact William at 
williamc...@umac.mo and/or visit
https://fhs.umac.mo/staff/academic-staff/williamchao/
https://williamchao.wixsite.com/williamchaolab

Applicants must hold a Ph.D. degree in relevant areas of structural biology or 
biological sciences.

What is the FHS?
The Faculty of Health Sciences at the University of Macau is a leading research 
and education institution in biomedical sciences. The Faculty aims to tackle 
the fast-evolving global health challenges through cutting-edge research and by 
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http://www.umac.mo/

What is Macau?
With over 300 years of Portuguese influence, Macau is a glittering 
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Mediterranean European culture. For further information, please visit
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https://en.wikipedia.org/wiki/Macau

[ccp4bb] Postdoc position in Integrated structural biology at EMBL Heidelberg, Germany.

[Pravinkumar Jagtap]

I am posting this on behalf of Dr. Janosch Hennig.
Regards,
Pravin

Location: Heidelberg, Germany
Staff Category: Postdoctoral Fellow
Contract Duration: 2 years
Grading: N/A
Closing Date: 29 October 2017
Reference Number: HD_01179

*Job Description*
The European Molecular Biology Laboratory (EMBL) is one of the highest
ranked scientific research organisations in the world. The Headquarters
Laboratory is located in Heidelberg (Germany), with additional sites in
Grenoble (France), Hamburg (Germany), Hinxton near Cambridge (UK), Rome
(Italy) and Barcelona (Spain).

EMBL Heidelberg is looking for a postdoctoral researcher to join the group
of Janosch Hennig at the Structural and Computational Biology Unit. The
candidate will work collaboratively with other members of the group on a
challenging project, involving several members of the TRIM protein family
and their involvement in development and disease. We try to understand the
connection between ubiquitylation and RNA binding mechanisms by using
integrated structural biology methods with a focus on NMR, but combining it
with X-ray and small-angle scattering (X-ray and neutron). Structural data
will be combined with proteomics and RNA-seq data to identify binding
partners, which further enables structural characterization of complexes to
ultimately obtain theories about their function in health and disease. The
successful candidate is expected to take a strong lead on his/her project
and start to develop independent ideas.

*Qualifications and Experience *
Experience with structural biology methods is required. Experience with NMR
and/or X-ray crystallography/small-angle scattering is beneficial as well
as lab and theoretical experience with proteinRNA interactions and general
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an interest in applied structural biology and biology in general.
Applicants lacking the above but have qualifications and expertise in RNA
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Experience in programming is not required but beneficial. The position
requires the ability to work collaboratively as this project is a group
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Please apply online through www.embl.org/jobs Additional Information EMBL
is an inclusive, equal opportunity employer offering attractive conditions
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Please note that appointments on fixed term contracts can be renewed,
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