Hi, I have a question about crude protein lysates (extracts). A fellow lab member in my lab is trying to isolate a 27KDa protein from here crude protein lysate. She mentioned that this is the smallest protein in her lysate (confirmed with SDS-PAGE). Right now, she is planning on running gel-filtration to isolate it but has yet to start. She did ask other labs at the school that have HPLC/UPLC about running her samples but it seems that something always comes up right before its time to run the sample. I mention that she could use a membrane 30KDa MWCO filter along with a vacuum filtration set-up to isolate her protein. I was curious if this is possible? She is ignoring my suggestions and I just want to know if its because my suggestion makes no sense. Any advice will greatly appreciated and will be passed on to my lab mate.
I would suggest to your fellow lab member that running a crude lysate down a gel filtration column is not a good idea. They are extremely expensive and generally used as a last purification step the remove minor contaminants from your protein of interest. A crude lysate contains thousands of proteins of different sizes and the peaks you would obtain on the HPLC would run into one another. It doesn't sound like this is a lysate from an over-expressed protein (in which case she would have cloned it into an expression vector that produces an affinity-tagged protein that would be easily purified with the appropriate affinity column), so the peak of her protein would easily be swamped by larger, broader peaks from more abundant proteins of a similar size.
I would suggest she should run a cation/anion exchange column first. These do not need an affinity tag and can be used to remove a large proportion of proteins that have an isoelectric point that is higher or lower than that of her protein of interest. After running this column (depending on the pI she could even run both a cation and an anion exchange column to get a higher purity) the sample should be less "dirty" and more suitable for gel filtration.
Regarding your own suggestion, I think you would end up using a large number of filters (they aren't exactly cheap) as they would clog up within seconds if you try to pass a crude lysate through them, and it would take you a day or two at least to filter the average volume of cell lysate (30 - 100 ml) this way. Even then, it would still contain all the contaminants that are smaller than 30 kDa ... and I find it strange that there are no proteins smaller that 27 kDA in her crude lysate.
I would suggest to your fellow lab member that running a crude lysate down a gel filtration column is not a good idea. They are extremely expensive and generally used as a last purification step the remove minor contaminants from your protein of interest. A crude lysate contains thousands of proteins of different sizes and the peaks you would obtain on the HPLC would run into one another. It doesn't sound like this is a lysate from an over-expressed protein (in which case she would have cloned it into an expression vector that produces an affinity-tagged protein that would be easily purified with the appropriate affinity column), so the peak of her protein would easily be swamped by larger, broader peaks from more abundant proteins of a similar size.
I would suggest she should run a cation/anion exchange column first. These do not need an affinity tag and can be used to remove a large proportion of proteins that have an isoelectric point that is higher or lower than that of her protein of interest. After running this column (depending on the pI she could even run both a cation and an anion exchange column to get a higher purity) the sample should be less "dirty" and more suitable for gel filtration.
Regarding your own suggestion, I think you would end up using a large number of filters (they aren't exactly cheap) as they would clog up within seconds if you try to pass a crude lysate through them, and it would take you a day or two at least to filter the average volume of cell lysate (30 - 100 ml) this way. Even then, it would still contain all the contaminants that are smaller than 30 kDa ... and I find it strange that there are no proteins smaller that 27 kDA in her crude lysate.
thank you for your reply. I forgot to mention that she did run it through a anion-exchange column prior to SDS-PAGE. Out of that, she has multiple proteins but her protein of interest is the smallest out of the rest. I just asked her and she stated that when she adjust the salt concentration to elute her protein of interest, the smallest protein out of all the proteins in the eluent is her protein of interest. Sorry about the confusion, I should restate that it is not really a crude lysate. Her plan is to do the gel-filtration next unless she can find someone to do the HPLC for her. Based on your suggestions, she should not run HPLC? Thank you for your suggestions and sorry for not revealing all the information.
Hi Anthony, I would suggest if possible get this protein cDNA and (re)-clone, overexpress it (if this is not the case) it with some His-tag and use IMAC first then anything else.
Andrei
Hi Anthony, If she has already run an anion exchange column then the sample should be clean enough for a gel filtration run. However, as I and Adrei mentioned earlier, it would be a hell of a lot easier to purify the protein if it were overexpressed with an affinity tag. Hence, I would also suggest she should clone the target gene from cDNA into a vector that adds an affinity tag to the protein (a His-tag or GST-tag are probably the most commonly used ones) for overexpression in a suitable host organism (an E. coli strain such as Rosetta2 would be my first choice). Affinity purification will give her a much cleaner sample that might not even need gel filtration depending on what she wants to do with the protein afterwards.
Thanks for all the replies it really is helping her. A little about her situation, she has cloned a holin gene specific towards a certain bacterium (cannot reveal) into a plasmid that has an arabinose inducer (araC). Yes, I agree, she should have thought it out more and included an affinity tag which she is now considering. Right now, she is using what she has because she will be attending the National ASM meeting and does not have time to start from scratch (It would not take her more than month in my opinion). I guess the gel-filtration method is better and now I know why she has ignored my suggestion about the MWCO filter. Does anybody have any suggestions regarding what type of sephadex she should use (order), medium or fine? I will forward your info to her and maybe she will take the advice and tag her protein in the future. Thanks again, especially to Dr. Ariza and Dr. Halavaty.
Hi Anthony, I think S75 column should be just fine for SEC purification step. Also, I would suggest when next time she runs IEC step, I assume she knows what ionic strength elutes this protein, so then she would need to hold the gradient at this ionic strength to get more pure protein, and then the protein is off the column continue linear or whatever gradient she is using. But she might already tried this. Anyway good luck! But I agree with cloning should not take more than 1 month.
Andrei
Hi Anthony, you have already received most of the possible solutions. Few more points. Though the size of the protein is 27 kDa, it might exist as an oligomer and therefore 30 kDa MWCO concentrator will still retain it. The purity of an untagged protein can also be enhanced by using a shallower gradient in ion-exchange chromatography. Also, adding another step of purification with a different type of column (such as cation-exchanger or hydrophobic interaction column) can help in removing some more contaminants.
Yes, ion exchange, followed by size-exclusion. That's the way to do it. Your filter is in effect a crude, low-resolution version of the size exclusion principle. It will result in some enrichment, but in practice will not give nearly as good resolution as a size-exclusion column.
Excellent suggestions from Anthony and Andrei. I think IEC is a good start point to begin a purification protocol. IEC is a versatil tool but some knowledge about the target and undesired proteins are needed (pI, pH range stability...) to obtain the best separation. Adequate pH in starting buffer and a fine slope (250 mM NaCl in15 Vc) in saline gradient for elution results in good separations. Gel filtration is recommended as the final step. I suggest to use Superdex 75 HR 10/30 column from GE Healthcare.
PI = 5.4
No stability range of pH but it works at pH 6 and probably up to pH 9.
I would first ask if your lab mate requires the protein in its native state i.e with its biological activity. The recommended way is first to separate your crude proteins according to their PI using ion exchange chromatography. This separates your crude proteins into 2; anaionic and cationic proteins and makes it much easier to purify. You will then need to check the differences in molecular weight of the proteins categorized with your protein of interest how high in molecular weight the next protein is to your protein of interest in PAGE. This will greatly assist you in designing how big your gel filtration column will be, bearing in mind that if the proteins molecular weights are so close, you will need a very long column to attain a separation. So you need a very clear procedure of determining your protein of interest. all the best
Update on results, she has ran the size exclusion chromatography and got inconclusive results. The void volume fraction has the same bands as the sample not passed thru the column. She is using biogel P30, with tris buffer at pH 7.6. The fractions after the void volume don't show any bands. This is her first try at this method. She is looking for any suggestions that might help her troubleshoot this issue. I am busy with my own project so I cannot provide any assistance to her except by passing along this information and fowarding any suggestions provided. Thanks
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