[RandomPerson]

I have a question related to DNA binding proteins.  The question is:

You want to purify a DNA binding protein that is involved in regulating oncogene expression in certain cancers.

a) What range of pI values would you expect for a DNA binding protein?

b) Name two different chromatographic techniques that you would use to purify this protein and provide a brief justification for your choices.

For a), I'm just a little confused as to how to determine that.  Would the pI value of the binding protein be around the DNA pI?  I'm just not sure how to approach this at all. 

For b), I'm thinking that the negative charge of DNA is important, and we talked about chromatography methods a lot in class, so ion-exchange chromatography makes sense to me for one choice, but I'm unsure as to whether gel filtration or affinity chromatography would the proper second choice. 

If you have any insight, let me know!

[yesway]

Hi,
For a), a premise for the exact DNA/Protein interaction is needed. Since pI is mentioned, I would guess Coulomb interaction.

DNAB-Proteins should have a pI at around 8-9 (or even higher? I'm not sure) to allow formation of favorable interactions at physiological pH.

b) IEC is an ok answer, AC with putative oligonucleotide interaction partner is an ok answer. SEC is also ok, but that is a kind of universal answer for protein purification exam questions (this is what it looks like ).

btw welcome to the world of protein scrubbing

best

[RandomPerson]

Thank you! 

[RandomPerson]

Actually, I have another related question on this topic. 

Basically, the problem talks about a protein that forms two distinct quaternary assembles (a homodimer and a homoheptamer) which do not interconvert.  It asks what is the best chromatographic technique to separate these two forms. 

My initial reaction was to say gel exclusion chromatography, but I feel like I'm missing something here. 

[Yggdrasil]

The sugar-phosphate backbone of DNA contains many negatively-charged phosphate groups.  Given this information, what types of residues do you think you would find on a DNA binding protein to facilitate interaction with the negatively-charged DNA?  What would these residues do to the pI of the protein?

For the homodimer vs homoheptamer separation question, gel filtration (size exclusion) chromatography is the way to go.

[RandomPerson]

Would you have positively charged residues like arginine and lysine?  Wouldn't they raise the pI of the protein?

[Yggdrasil]

You are correct.

[Babcock_Hall]

Would you have positively charged residues like arginine and lysine?  Wouldn't they raise the pI of the protein?

So how could you use the elevated pI of your protein to help you separate it from a protein with pI of, say, 6?

[RandomPerson]

Would you have positively charged residues like arginine and lysine?  Wouldn't they raise the pI of the protein?

So how could you use the elevated pI of your protein to help you separate it from a protein with pI of, say, 6?

Are you asking for a technique here?  Isoelectric focusing is the only one that I know of that separates based on pI.

[Babcock_Hall]

You can use either cation exchange chromatography or anion exchange chromatography to separate proteins on the basis of charge.  The net charge of a protein depends on two factors, pI and pH, and the latter is under the control of the experimenter in his or her choice of buffer.