Re: [ccp4bb] co-express two proteins in E.coli
We have also had success expressing two proteins separately and then mixing the soluble fractions of the lysates in the presence of a complex-initiating ligand, where one protein has a His-tag (preferably the protein that expresses to a lower extent) and the other no affinity tag followed by a Nickel affinity column to capture the complex (unpublished). Laurie Betts UNC Chapel Hill On Mon, Jul 16, 2012 at 11:55 PM, Biswajit Pal p...@ccmb.res.in wrote: I fully agree with Dima. We are able to co-express and purify two interacting partners using pET28 and pET21 in E. coli. Some related references are : J. Mol. Biol. (2011) 405,49–64 J. Biol. Chem. (2006) 281, 26491–26500 J Struct Biol. (2011) 175(2):159-70 Biswajit - Original Message - From: Dima Klenchin klenc...@facstaff.wisc.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 17, 2012 5:16:34 AM Subject: Re: [ccp4bb] co-express two proteins in E.coli I have been using the Duet system from Novagen (or whatever it is called these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins did not work in either vector. Either, one protein expressed or the other. I played around with the promotors (they are both T7) by changing one to the tac promotor. This increased the expression of this gene but shut off expression of the other. The only way I could get my proteins to co-express was to use pGEX vector with one protein, and pRSFDuet with the other protein (leaving the second MCS empty). There is a paper which sums up co-expression in E coli. http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-55.html There is really absolutely no problem co-expressing two proteins both from near-identical pET vectors as long as the two plasmids carry difference selection marker. We've used pET24 in combination with pET28 or pET31 on several complexes and it always works as long as you keep both antibiotics around. - Dima
[ccp4bb] co-express two proteins in E.coli
Dear ALL, We are planning to co-express two proteins in E.coli. Could anyone suggest a good dual set plasmid or a proper insertion sequence between two genes, including the Shine-Dalgarno sequence? Thank you very much and have a nice summer. Jerry McCully
Re: [ccp4bb] co-express two proteins in E.coli
I've had success with pet-Duet. http://ecoliwiki.net/colipedia/index.php/pETDuet-1 Jason. -- Jason Busby PhD Student Laboratory of Structural Biology School of Biological Sciences University of Auckland Thomas Building 110 3a Symonds St Private Bag 92019 Auckland 1142 New Zealand ph: +64 9 3737599 ext 84155 fx: +64 9 3737414 On 17/07/2012, at 9:23 AM, Jerry McCully wrote: Dear ALL, We are planning to co-express two proteins in E.coli. Could anyone suggest a good dual set plasmid or a proper insertion sequence between two genes, including the Shine-Dalgarno sequence? Thank you very much and have a nice summer. Jerry McCully
Re: [ccp4bb] co-express two proteins in E.coli
I have been using the Duet system from Novagen (or whatever it is called these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins did not work in either vector. Either, one protein expressed or the other. I played around with the promotors (they are both T7) by changing one to the tac promotor. This increased the expression of this gene but shut off expression of the other. The only way I could get my proteins to co-express was to use pGEX vector with one protein, and pRSFDuet with the other protein (leaving the second MCS empty). There is a paper which sums up co-expression in E coli. http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-55.html Dan
Re: [ccp4bb] co-express two proteins in E.coli
I have been using the Duet system from Novagen (or whatever it is called these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins did not work in either vector. Either, one protein expressed or the other. I played around with the promotors (they are both T7) by changing one to the tac promotor. This increased the expression of this gene but shut off expression of the other. The only way I could get my proteins to co-express was to use pGEX vector with one protein, and pRSFDuet with the other protein (leaving the second MCS empty). There is a paper which sums up co-expression in E coli. http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-55.html There is really absolutely no problem co-expressing two proteins both from near-identical pET vectors as long as the two plasmids carry difference selection marker. We've used pET24 in combination with pET28 or pET31 on several complexes and it always works as long as you keep both antibiotics around. - Dima
Re: [ccp4bb] co-express two proteins in E.coli
I fully agree with Dima. We are able to co-express and purify two interacting partners using pET28 and pET21 in E. coli. Some related references are : J. Mol. Biol. (2011) 405,49–64 J. Biol. Chem. (2006) 281, 26491–26500 J Struct Biol. (2011) 175(2):159-70 Biswajit - Original Message - From: Dima Klenchin klenc...@facstaff.wisc.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 17, 2012 5:16:34 AM Subject: Re: [ccp4bb] co-express two proteins in E.coli I have been using the Duet system from Novagen (or whatever it is called these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins did not work in either vector. Either, one protein expressed or the other. I played around with the promotors (they are both T7) by changing one to the tac promotor. This increased the expression of this gene but shut off expression of the other. The only way I could get my proteins to co-express was to use pGEX vector with one protein, and pRSFDuet with the other protein (leaving the second MCS empty). There is a paper which sums up co-expression in E coli. http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-55.html There is really absolutely no problem co-expressing two proteins both from near-identical pET vectors as long as the two plasmids carry difference selection marker. We've used pET24 in combination with pET28 or pET31 on several complexes and it always works as long as you keep both antibiotics around. - Dima
