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<mailto: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<mailto: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 <rafael_mmsi...@hotmail.com> 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<mailto:kelvin....@epfl.ch>
Enviado:quarta-feira, 8 de julho de 2020 16:22
Para: CCP4BB@JISCMAIL.AC.UK<mailto: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
<umarfaroo...@gmail.com<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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