Re: [ccp4bb] Expression of a protein of 43KDa

...@jiscmail.ac.uk] On Behalf Of Kerff Frédéric Sent: 18 February 2010 23:16 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Expression of a protein of 43KDa The maltose binding protein is one possibility. Some collaborators did provide us with very pure MBP instead of the protein of interest... Mass spec

Re: [ccp4bb] Expression of a protein of 43KDa

Dear Armando, Two similar, but not identical experiences: - we expressed a protein and got some ugly small crystals. Then the Edman degradation results on the purified protein came in and turned out to correspond to chloramphenicol transferase (same size as our protein). - expressed a

Re: [ccp4bb] Expression of a protein of 43KDa

EF-Tu Poul On 18/02/2010, at 18.49, Armando Albert de la Cruz wrote: I am trying to overexpress a His-tagged protein of 29KDa in E.coli (BL21-codon plus) and I end up with a highly expressed product that of 43KDa that binds to the Ni-column. I also have nice crystals. Does anyone have any

Re: [ccp4bb] Expression of a protein of 43KDa

Maybe I am not getting it, but I don't see your problem or question? What I understand from your message is that you have a protein that overexpresses, purifies, and crystallizes... where's the problem? We all wish we had that problem... Jon On 02/18/2010 11:49 AM, Armando Albert de la

Re: [ccp4bb] Expression of a protein of 43KDa

496 3792 Lab -Original Message- From: Jon Schuermann [mailto:schue...@anl.gov] Sent: Thursday, February 18, 2010 4:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Expression of a protein of 43KDa Maybe I am not getting it, but I don't see your problem or question? What I understand

Re: [ccp4bb] Expression of a protein of 43KDa

Well that it's not his protein which he has crystallized but an over expressing contaminant :-) Or the alternative would be although it's supposed to be 29kDa it runs funny and appears larger in the SDS gel, in that case it would be OK. If we only had some confirmation that the 43kDa band is

Re: [ccp4bb] Expression of a protein of 43KDa

I see the issue now, sorry... I agree with Jurgen. Did you get it DNA sequenced before overexpression? Size of tag? Jon On 02/18/2010 03:06 PM, Jürgen Bosch wrote: Well that it's not his protein which he has crystallized but an over expressing contaminant :-) Or the alternative would be

Re: [ccp4bb] Expression of a protein of 43KDa

The maltose binding protein is one possibility. Some collaborators did provide us with very pure MBP instead of the protein of interest... Mass spec eventually identified it after we wasted some time on it... Fred I am trying to overexpress a His-tagged protein of 29KDa in E.coli (BL21-codon