Has anyone expressed and purified proteins with basic PI? I am purifying a protein which has a PI of 8.78. I see a band on SDS-PAGE when protein and solubility tag are together. However, After cleaving the tag, my protein becomes highly unstable and i do not get anything after gel filtration or ion exchange chromatography. Is there any way to enhance the stability of proteins having basic PI?
Thanks,
Sunil Singh
protein instability may have nothing to do with the basically of the protein: Histones are highly basic and highly soluble proteins. your protein may be misfolded - either due to the tag or simply because it is misfolded on the conditions you are using. your protein may adhere to certain types of plastic tubes (DNA LoBind are notorious), your protein may favor certain buffer conditions (MBP percipiates upon introduction of ion-pairing additives).
I would suggest you run a controlled experiment where you check whether you lost your protein on any stage from cleavage onwards.
Hi there,
What tag are you using? Some act as solubilizing agent or chaperon on your favorite protein (MBP , GST). It might be that cleavage of the tag is inducing destabilization. And as David mentioned there is little to do with the fact your protein is basic. What may be important is the pH at which you actually work: if too close to pI it may affect protein stability.
Dear All,
Thank you for all your suggestions. I am using MBP solubility tag. My protein is stable however, it aggregates with tag or after the cleavage of the tag. Literature suggests that my protein could exist as a dimer. I have also tried SlyD tag. I get soluble protein but according to DLS readings its a homogeneous aggregation.
I am using HEPES buffer (pH 7.0)+400mM Nacl+0.5M Arg+20mM MgCl2 in my lysis buffer. i also add small amount of Triton X100.
What else can i add to suppress aggregation? Can i try this protein (homogeneous aggregate) for crystallization?
Thanks & Regards,
Sunil Singh
there are two things you can do to reduce aggregation:
1. reduce concentration of your protein, and
2. use adequate buffers.
there is no predetermined conditions for protein solubility and you have to check different buffer conditions. maybe your protein is losing solubility in pH 7 and prefers a more acidic pH. or more basic one. maybe there is too much salt in your buffer, or too little. maybe you need an ionic detergent, maybe non-ionic. maybe you need it to be under the CMC concentration and maybe above. you really have to run different conditions and see.
Cheers,
David.
Need some clarification of your experiments: Is the expression system bacterial or eucaryotic? Does the protein have cysteines? Your criteria for stability is DLS only? When you write "My protein is stable however" in your second note are you referring to the literature or your sample? If DLS shows a homogeneous aggregate then are you indicating that all the aggregate molecules are identical and therefore have the same number of constituent monomers and mass? How do you generate the feedstock that goes onto the affinity column? Just after the affinity column you do SDS-PAGE and see monomer mass of the tagged protein but you didn't write about what was seen on SDS gels after cleavage. Were the PAGE gels run both reduced and non-reduced? The SDS is denaturing so you won't see non-covalent aggregates, just covalent ones in the absence of reducing agents. From your second note, you claim that after the affinity purification the protein is aggregated whether or not MBP tag is present so this must be from DLS assay but not from SDS gel assay, right? You have to use DLS because you can't get chromatography by any modality, right? The protein is precipitating at the head of the column or binding to the inlet frit so if you strip your columns with 4-6 M guanidine HCl you might get material balance. This would be a classical case of mis folded protein behavior that I've experienced and obviously David as well. Can you get any reverse phase chromatography to work? You'll need LC/MS anyway to confirm amino acid sequence because there may be a mutation regardless of how perfect the DNA sequencing is. A broad peak in reverse phase chrom would also indicate misfold as well as the shape of the band on SDS-PAGE. Until you know your protein is properly folded it may be futile to eliminate aggregation.
If you're lysing bacterial cells then type 1, type2, or enzymatic cell breakage can make a difference. If this is a eucaryotic protein with cysteines then you already know that the cysteines will be reduced in a bacterial environment. I assume this is solubly expressed because you make no mention of refolding and likely the cysteines (if present) will need to properly pair and oxidize.
Yes, we have done this many times in our lab. The most basic protein (mAB) I have worked with had a pI of around 9.9. I have attached for you a poster, where you can see the separation of this protein into its isoforms (middle of the poster).
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