Dear people,
I just would like to hear your opinion on the posible meaning of the next observation.
I am presently trying to purify a protein that is suspected to assemble with other protein partners in cells. The recombinant histagged protein is well expressed in E coli, and is indeed purified with moderate yields (about 1mg/L). However much material is lost as insoluble, after lysis.
The reason why I decided to write is that other bands are often accompanying the purified protein in SDS-PAGE gels. Surprisingly, the most intense bands were found by MS analysis to correspond to DnaJ, DnaK, GroL, IpbA and IpbB chaperones (shown for protein 1 in the attached image). One possibiity is that these chaperones were copurified bound to the target histagged protein (revealing an assistance to the protein, presumably improperly folded or lacking a cofactor ). However, contaminating bands are relatively intense (yet, variable from experiment to experiment).
What do you think about such possibility ? I have purified many proteins, and lost many cases due to insolubility and formation of inclussion bodies, but I cannot remember of any such case. One example is for instance protein2 in the attached image.
Alternatively, could this observation reveal a potential chaperone role of "my protein" ?
Any help would be really appreciated.
Best regards,
Luis
We have found chaperons co-purifying with poorly folding proteins- wash with same buffer containing 400 mM KCl, 2.5 mM MgCl2 and 1 mM ATP to get rid of E.coli chaperones.
Hi,
I agree with Annemarie. In some cases not only poorly folding proteins will be attached to chaperones, but over expressed ones too. If you are loosing most of your protein in pellet, why not to stop cell grow at lower level with fewer expressed protein.
Good luck!
The contaminated proteins you listed has excellent MS detection signal, from trypsin digestion to charged species for flying in the mass detector.
You may also try E.coli systems with extra chaperons for protein folding and express your protein in lower Temp such as 16c.
Yes, chaperones are extremely common contaminants. They are triggered by the stress of recombinant overexpression and production of poorly folding or insoluble proteins. They also tend to copurify as well as stick to chromatography resins. There are many papers describing this in the protein purification and proteomics literature.
Thank you !
I therefore consider as most likely the possibility that I got contaminants that assisted the correct folding of my protein.
I would appreciate anyway if Ho Leung could indicate one of the references he mentions, and most importantly if Haijung Liu could tell me where to get such E coli strains with extra chaperons from.
Thank you again !
Chaperone plasmid set
http://www.clontech.com/BT/Products/Protein_Expression_and_Purification/Bacterial_Expression_Systems/Protein_Folding/Chaperone_Plasmid_Set
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