[ccp4bb] RES: [ccp4bb] RES: [ccp4bb] Looking for suggestions with protein expression

[Rafael Marques]

Thank you for pointing me this out. I don’t know why I said BL21...maybe in the 
rush to say to use Rosetta for protein expression. We use Dh5a for plasmid 
replication (although I have already used BL21 for plasmid purification and it 
worked nicely). My bad.


Rafael Marques da Silva
Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"


De: bogba...@yahoo.co.uk
Enviado:sexta-feira, 10 de julho de 2020 00:30
Para: Rafael Marques
Cc:CCP4BB@JISCMAIL.AC.UK
Assunto: Re: [ccp4bb] RES: [ccp4bb] Looking for suggestions with protein 
expression

Re:  "In our lab we generally use BL21 for plasmid replication."

I am interested because on the occasions that I prepared plasmids in BL21 
(usually by mistake), they were never any good for cloning or sequencing. I 
always had to transform them back into a cloning strain and do another plasmid 
prep! Probably my incompetence!
Jon Cooper

On 9 Jul 2020 21:44, Rafael Marques  wrote:

Hi Umar,



I must say that it would be better use as an expression system Rosetta DE3 or 
Rosetta-gami instead of BL21. In our lab we generally use BL21 for plasmid 
replication.

Also, there are a few things that you could try before trying another 
construction.



  1.  Lower the temperature during the expression.
  2.  Try to use a different range of pH in your buffer. Maybe you could add a 
bit of NaCl (150 – 300 mM) and/or glycerol (2 – 10%)
  3.  I must say that I have already obtained very different results using Co 
or Ni columns for IMAC. You could take a look at this.



Regards



__



Rafael Marques da Silva

Mestrando em Física Biomolecular

Universidade de São Paulo



Bacharel em Ciências Biológicas

Universidade Federal de São Carlos



phone: +55 16 99766-0021



   "A sorte acompanha uma mente bem treinada"





De: Lau Kelvin
Enviado:quarta-feira, 8 de julho de 2020 16:22
Para: CCP4BB@JISCMAIL.AC.UK
Assunto: Re: [ccp4bb] Looking for suggestions with protein expression



Hello Umar,



I would not pin down your difficulties solely due to an Fe-S proteins. I have 
produced some with no fusion partners and they work wonderfully. They were 
expressed in an aerobic environment and then reduced in an anaerobic one before 
usage in reactions.



1) On the Fe-S side, there are plasmids you can co-transform to increase Fe-S 
production. This plasmid pH151 has the synthetic genes necessary for Fe-S 
formation.

https://www.jbc.org/content/279/33/34721.abstract



2) On the general protein side, have you hhpred your protein? Different 
constructs (not just tags), temperature? Strain? Media?



3) For these proteins we typically use His Excel (or Protein Ark Ni2+ Advance) 
that is resistant to most chelators since more often than not, they contain 
other metals and can snatch Ni2+ from normal Ni-NTA resins. Strep tags also 
work well.



On Jun 27, 2020, at 9:14 AM, Umar Farook 
mailto:umarfaroo...@gmail.com>> 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.







--

Best Regards,

Umar Farook





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[ccp4bb] Fully funded PhD position

[Jesko Koehnke]

Dear All,

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The position is available from Oct 1st, but later starting dates are of
course possible.

Applications will be reviewed on a continuous basis until the position is
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Re: [ccp4bb] RES: [ccp4bb] RES: [ccp4bb] Looking for suggestions with protein expression

[Jon Cooper]

Hello Rafael

No problem, thank you for confirming.

Best wishes

Jon Cooper

 Original Message 
On 10 Jul 2020, 14:27, Rafael Marques wrote:

> Thank you for pointing me this out. I don’t know why I said BL21...maybe in 
> the rush to say to use Rosetta for protein expression. We use Dh5a for 
> plasmid replication (although I have already used BL21 for plasmid 
> purification and it worked nicely). My bad.
>
> Rafael Marques da Silva
>
> Mestrando em Física Biomolecular
>
> Universidade de São Paulo
>
> Bacharel em Ciências Biológicas
>
> Universidade Federal de São Carlos
>
> phone: +55 16 99766-0021
>
> "A sorte acompanha uma mente bem treinada"
> 
>
> De: bogba...@yahoo.co.uk
> Enviado:sexta-feira, 10 de julho de 2020 00:30
> Para: [Rafael Marques](mailto:rafael_mmsi...@hotmail.com)
> Cc:CCP4BB@JISCMAIL.AC.UK
> Assunto: Re: [ccp4bb] RES: [ccp4bb] Looking for suggestions with protein 
> expression
>
> Re:  "In our lab we generally use BL21 for plasmid replication."
>
> I am interested because on the occasions that I prepared plasmids in BL21 
> (usually by mistake), they were never any good for cloning or sequencing. I 
> always had to transform them back into a cloning strain and do another 
> plasmid prep! Probably my incompetence!
>
> Jon Cooper
>
> On 9 Jul 2020 21:44, Rafael Marques  wrote:
>
>> Hi Umar,
>>
>> I must say that it would be better use as an expression system Rosetta DE3 
>> or Rosetta-gami instead of BL21. In our lab we generally use BL21 for 
>> plasmid replication.
>>
>> Also, there are a few things that you could try before trying another 
>> construction.
>>
>> -  Lower the temperature during the expression.
>> -  Try to use a different range of pH in your buffer. Maybe you could add a 
>> bit of NaCl (150 – 300 mM) and/or glycerol (2 – 10%)
>> -  I must say that I have already obtained very different results using Co 
>> or Ni columns for IMAC. You could take a look at this.
>>
>> Regards
>>
>> __
>>
>> Rafael Marques da Silva
>>
>> Mestrando em Física Biomolecular
>>
>> Universidade de São Paulo
>>
>> Bacharel em Ciências Biológicas
>>
>> Universidade Federal de São Carlos
>>
>> phone: +55 16 99766-0021
>>
>> "A sorte acompanha uma mente bem treinada"
>>
>> 
>>
>> De: [Lau Kelvin](mailto:kelvin@epfl.ch)
>> Enviado:quarta-feira, 8 de julho de 2020 16:22
>> Para: CCP4BB@JISCMAIL.AC.UK
>> Assunto: Re: [ccp4bb] Looking for suggestions with protein expression
>>
>> Hello Umar,
>>
>> I would not pin down your difficulties solely due to an Fe-S proteins. I 
>> have produced some with no fusion partners and they work wonderfully. They 
>> were expressed in an aerobic environment and then reduced in an anaerobic 
>> one before usage in reactions.
>>
>> 1) On the Fe-S side, there are plasmids you can co-transform to increase 
>> Fe-S production. This plasmid pH151 has the synthetic genes necessary for 
>> Fe-S formation.
>>
>> https://www.jbc.org/content/279/33/34721.abstract
>>
>> 2) On the general protein side, have you hhpred your protein? Different 
>> constructs (not just tags), temperature? Strain? Media?
>>
>> 3) For these proteins we typically use His Excel (or Protein Ark Ni2+ 
>> Advance) that is resistant to most chelators since more often than not, they 
>> contain other metals and can snatch Ni2+ from normal Ni-NTA resins. Strep 
>> tags also work well.
>>
>>> On Jun 27, 2020, at 9:14 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.
>>>
>>> --
>>>
>>> Best Regards,
>>>
>>> Umar Farook
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
> --

[ccp4bb] unsubscribe

[Israel Sanchez]

-- 
Israel S. Fernandez PhD
Ramakrishnan-Lab
MRC Laboratory of Molecular Biology,
Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.



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