[ccp4bb] B-factor distributions

[Robbie Joosten]

Posted on behalf of Gert Vriend:

This article didn't make it to Nature Methods (...), but might be of interest 
to theoreticians interested in B-factor distributions:
 https://journals.calstate.edu/pump/article/view/2409/2168 

Gert Vriend



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

[Manjula Ramu]

Hi,

I agree with Rafael. Using of glycerol for aggregating proteins works
usually. Try also purifying in range of buffers (of course considering the
pI of the protein). A lower temperature may reduce the yield but worth a
try if it is aggregating at higher concentration.
You can also try precipitating other contaminants at their pI, before the
affinity purification steps (by dialysing the lysate).


Thanks and Regards,

Manjula Ramu






On Fri, Jul 10, 2020 at 11:46 AM Jon Cooper <
488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello Rafael
>
> No problem, thank you for confirming.
>
> Best wishes
>
> Jon Cooper
>
>
>
>
>
>  Original Message 
> On 10 Jul 2020, 14:27, Rafael Marques < rafael_mmsi...@hotmail.com> 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 
> *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  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