My I initial thought is your protein concentration that you load onto the column is now higher in the 16 liter prep. Then the equilibrium shfits to the aggregated form. The aggregation usually is protein concentration dependent. So I would check what the mg/ml was in the 4 liter prep when you loaded the column and use the same amount. I would also use the same volume you used in the first experiment.

0.1 M NaCl in the buffer will increase the solubility of your protein if it is intrinsically soluble. Alternatively, if the protein has hydrophobic nature being a membrane protein then a mild detergent (like 0.1 % triron X-100 ) will help. As a rule, for membrane protiens detergents are used during all the purification steps

Dear Niyaz, if the detergent is used with membrane proteins, the purification with sizing columns becomes difficult. This is because, first that micelles could form, and other proteins get into the micelles. In addition, the protein does not come out as a monomer, but as an aggregate as a broad peak. This is the typical situation I have found. But maybe others have had success with this. Adding salt with a hydrophobic protein will increase the aggregation likely, but adding glycerol or sucrose may help.

If the detergent is lower than the CMC, it may work well. I just had no success when I tried the size exclusion column with my membrane protein.

Hi

a couple of questions, which detergent are you using and which type of concentrator are you using, also how did you scale the solubilisation from 4 to 16L did you maintain the same protein to detergent ratio?

The buffer I am using is 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.075% C10E5. And I solubilize the protein in 1% DDM. I use 50kDa cut-off concentrator from Millipore. I didn't do anything to upscale the solubilization process, because the yield of the protein is really low, less than 0.1mg/L culture. So I don't think the aggregation problem is due to the large amount of protein...

A couple of things to keep in mind, poor expression will always make things difficult especially with membrane proteins as this will cause problems with solubilisation due to volume issues. The first step is to get the best membrane preparation you can so bust the cells as best you can, we use a microfluidizer, then you may want to wash the membranes with salt (typically between 500-1M NaCl). The next step is to optimise the solubilisation, for structural work we use the Anatrace DDM (the best quality we can buy) Membrane proteins are typically extracted by detergent concentrations of 0.5-2.0%, using detergent/protein ratios of 1:1 to 3:1. So as you scale up you have to dilute your membrane prep back to get the 3:1 ratio, this can make loading the column increasingly difficult as you have larger and larger volumes (to get around this we either use batch IMAC although this has limits or use a StrepII tag which has allowed us to work with >1L of solubilised lysate) If you don’t obtain a good solubilisation efficiency then the ratio of aggregate to protein of interest is poor as you dont extract enough of your protein. The other thing to check in your SEC is the agg peak protein (yours) or DNA or some high MW protein that is just causing scatter at A280. When using C10E5 this has an aggregation number of ~73 so will have an MW of around 25KDa so a 50KDa MWCO concentrator should be ok (DDM tends to stick to protein so you may carry some through), but when concentrating resuspend every 5mins between spins so as to reduce detergent concentration. If you concentrate up significant amounts of detergent this can destabilise the membrane protein, also keep in mind you need to minimise the elution volume from the IMAC as the larger the volume the more detergent you will concentrate.

For the type of membrane proteins I work with, I focus on how to thermostabilise the protein as best I can. I am in a good position as some of this work is done through mutagenesis however you can gain a significant amount of thermostabilisation through addition of CHS (lipids) Ligands and adjusting salt and buffer conditions, glycerol all of this will help keep your protein happy and increase your yield. Finally look at the detergent you are using C10E5 may be too harsh for your protein and be causing the losses you see if you are planning vapour diffusion work then you need to minimise the size of the micelle if you are planning LCP then this isnt a real issue as the detergent is only a way to get the protein into the lipid phase so working with DM or DDM is fine.

Dear James, I am really grateful to your answer. I will explain in a more detailed way.

I am also using microfluidizor to break cells, and the solubilization conditions are also very wimilar with yours. As for the expression level problem, I have tried to optimize, like different media, temperature, promoter, inducer concentration and inducing time. However, none of these hepled. Maybe the reason of low yield is that I express the gene from a hypothermophilic bacteria in E.coli.

I have tested the agg peak in SEC with SDS PAGE, and it shows no difference as the main peak and is my target protein, this has been confirmed with MS. And if I take the agg peak, concentrate it and load on an analytical superdex200 column, I can still get two peaks: an agg peak and a normal one, which means, the aggregation is a reversible process.

As for the C10E5 question, actually, I have got initial crystals in this detergent, that's why I am sticking to this one. Moreover, I have tried a couple of other detergents, even in DDM, it still has this upscaling problem...

You seem to be an expert in membrane protein crystallization, as a beginner, I feel honored to discuss with you.

Thank you!

Dear Rhine, the aggregation could occur even at lower protein concentrations. My thought was that in the 16 liter prep you had changed the conditions since it worked quite well for you in the 4 liter prep. Since the aggregation is reversible and you may not want to use glycerol since you are trying to crystallize the protein, I would use a more dilute sample. Aggregation is concentration dependent and it could occur at low protein concentrations. But your problems are not unique given that you have a membrane protein and it is in detergent.

If the aggregation is occurring on the column, it is likely occurring when you form crystals, so if you don't want this to happen, you may try to change conditions of the buffer and detergent as James has suggested.

My other question is what does it matter if it aggregates on the column? Are you using this as a purification step or buffer exchange. Once you get it off the column, if you concentrate it, it would aggregate again. So unless you change the buffer, you will have this problem. But it may not be a problem, if you get crystals, and the structure is meaningful. The protein may exist in a multimeric state in Vivo.

Thank you all for your suggestions.

Actually I have screened for the buffer condition of purifying this protein, and of course I did this in small scale. I cannot screen conditions using 16L culture each time...The detergent and pH I am using now is the best I can get...

Dear Rhine, my suggestion would be to do a simple protein assay and adjust your solution accordingly so that everything is reproducible.

The problem could be at expression level. If your proteins are in "inclusion bodies" , then one can expect a lot of S-S bonds. In similar situation I have used reducing agents [mercapto compounds] followed by 4-8 M urea extraction [you will have standardize the concentration of urea at witch you proteins gets released from the inclusion bodies

From what you have said it sound like you have a very tough target, typically when we arrive at this position we tend to look at changing the construct in some way, for GPCR's this tends to be quite simple N, C and IC3 loop truncations and the introduction of fusion parteners. This is screened though a combination of FSEC and radioligand binding/thermostabilisation assays. Alternatively you can look for homologues of your protein, I have seen a protein not being expressed at all changing to a very well expressed protein. Another potential direction is to follow on form a presentation I saw from a member of David Drews group they were able to improve the expression of bacterial membrane proteins significantly using MemStar approach this was really amazing

http://technology.sbkb.org/portal/list/technology/Protein%20Expression/pagename/?page=6