Hi

"I wonder if I could add 1mg/ml protein1 into my protein(7-10mg/ml) to induce it crtstal? "

Probably not, but won't know unless you try it.

"Any other suggestion can be tried to acquire crystals of protein2?"

change protein buffer

change purification strategy

purifying the ligand complexes (e.g. gel filtration)

add more native ligands (make it bigger)

limited proteolysis (make it smaller)

reductive alkylation

mutagenesis

expression in a different system

->  all of the above in any possible combination ?

"Can I add protein 1's crystal into protein 2, to help protein2 to nucleate? like seeding?"

Probably not, but won't know unless you try. If the proteins 1 and 2 are closely related (e.g. very high sequence identity) this approach would be a standard procedure.

good luck, crystallization can take time

Hi, Stefan. Thank you for your suggestion!

About your advice:

Dynamic Light Scattering has been done to test a relatively good buffer condition make protein2 as monomer.

After nickel column affinity, ion exchange chromatography and gel filtration were used to purified protein2. SDS-PAGE shows >95% purity of protein2. 

Cloned into some plasmid, like pRSF with sumo tag, protein2 would break into N-ter and C-ter. Gel filtration can't seperate the full length from the two terminal domain. I have been mutating the amino acid around the broken region. Yet proteolysis can cut protein2 into two domanis.

Protein1 is not the homologous protein of protein2, but they have interaction. I aslo wanna try my idea.

Yesterday I run native-page, I first put it into EB strain, and protein2 have fluorescence under UV light. This phenomenon shows protein2 could twine with nuleotide. R250 shows, simultaneously, that was protein2. Do you have some advice for me to seperate nuleotide from protein?

I don't understand 'native ligands' and 'ligands complex' really mean. Dose 'native ligands' relate to membrane proteins, like GPCR?

Thank you for your encouragement. :D

"Gel filtration can't seperate the full length from the two terminal domain."

How about Ion exchange or affinity chromatography (if applicable)?

" I have been mutating the amino acid around the broken region. Yet proteolysis can cut protein2 into two domanis."

Since you tried to crystallize the full length protein, recover some of the old drops from various conditions and run them on a gel and see if your protein cleaves the same way under crystallization conditions.

" Protein1 is not the homologous protein of protein2, but they have interaction. I aslo wanna try my idea." 

In that case protein 1 is  a ligand of protein 2, you should try to purify both together instead of just mixing them by an arbitrary ration. Also, repeat the limited proteolysis with the purified complex of full length proteins 1 & 2.

"I first put it into EB strain, and protein2 have fluorescence under UV light. This phenomenon shows protein2 could twine with nuleotide."

Technically, you showed that something in that band can bind EB stain. Have you measured the 260:280 ratio in that sample?

"Do you have some advice for me to seperate nuleotide from protein?"

- Polyethyleneimine precipitation

- High salt during affinity purification

- DNA cellulose chromatography

- Heparin Sepharose chromatography

- Don't remove it if it keeps the protein stable

"I don't understand 'native ligands' and 'ligands complex' really mean"

native ligands would be protein 1 or RNA(?) or ATP or whatever else binds naturally to your protein. You might want to use non-natural ligands, like non-hydrolysable  ATP mimics, Anti-bodies against the protein1:2 complex, detergents, lipids...any combination of those might help you getting crystals.

good luck,

S.

Hi, Stefan.

I have got Ion exchange chromatograhy before gel filtration, and there are two peaks. Peak1 occured at 210mM NaCl concentration. Peak2 occuerd at 250mM NaCl concentration. Peak1 is much purifier than peak2. But also twined with proteins cleaved into two domain of clone having no tag. With 10×His tag, the cleavage is lesser. The clone with tag are more stable.

I absorbed crystal spots both tag-clone and no-tag-clone to run SDS-PAGE, and I found both of them still have protein1; no-tag protein1 still contains the two domain and the full length one(don't totally cleave into two domain).

I have directly added 1mg/ml protein2 into protein1(7mg/ml, 200ul) to crystal screen. Wait. Observe mostly conditions are turbid and bronw. I will try to get a gel filtration after adding protein1 and protein2 together. Do I have to ensure they are the same charge property? At pH7.5, protein1 is negative charge, protein2 is inverse.

Protein1: A260/280=1.2 shows there really are nuceonides twining protein1. I have tried using 200mM NaCl; 100mM MgCl2; 100mM MnCl2; RNase and DNase. T.T~~~ These extreme buffer conditions are cruel to protein1. Protein1 were all in precipitant.Would you like to sopport some buffer formula for me?

The pI of protein1 is about 6.0. If I wanna use Heparin, should I use pH under 6.0? otherwise protein1 wouldn't combine with the column. Use sodium citrate?

thouk you~~~>.

Hi, if I understand correctly you are asking about a seeding procedure (with protein1) to induce the nucleation/crystallization of protein2. This is a possibility, but the chances are best when protein1 and protein2 are very homologous - but even then there is no guarantee.

If they are very homologous, you will need to crystallize protein1, and use the crystals (or fragments thereof) as seeds in crystallization setups of protein2. In my opinion it doesn't make sense to introduce a protein1 solution together with protein2.

You can try. It's called cross-seeding. Best results can be obtained if you cross seed crystallization screens rather that the fine tuned optimization grids for protein 1. It works mostly when both proteins have similar structure. Seeding full-length protein with crystals of its single domain was done succesfully in some cases as well. It's definiatelly worth trying.

But you should follow seeding protocol., which I haven't made clear.