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For extracting Lysozyme from egg white which method would you recommend and please help me understand why.

Lysozyme has a higher isoelectric point (pH 10.5) and a lower molecular mass (14 307 Da) than most proteins so both these methods make quite a bit sense. Use Sephahdax G100 for gel permeation chromatography and CM-Sephadex G25 ion exchanger for ion exchange.

From what I can understand, Gel Permeation will separate the egg white based on Mr of the various proteins and compounds. There is quite a bit of variation in the Mr of egg white proteins but lysozyme, in fact, doesn't have the lowest Mr as Cystatin and Ovomucoid have lower Mr. Furthermore, GPC requires around at least a 10% difference in molecular weight for a reasonable resolution of peaks to occur. The range of Mr is also quite broad in this method. Will this be disadvantageous perhaps? So GPC does have the plus side of it having a well-defined separation time due to the fact that there is a final elution volume for all unretained analytes.

Now Ion exchange chromatography is very specific in the nature of how the exchanger interacts with the mixture being separated. Lysozyme has a PI of 10.7 which is much higher than the rest of the proteins in the egg white. As with GPC we use 0.05M Tris-EDTA, pH 8.2, containing 0.05M NaCl as the standard. Can this setup result in Gibbs Donan effect or chromato- focusing ?

I'm currently leaning towards GPC as the option to yield the purist product especially seeing as it can be desalted afterwards with G25.

Please help guide me in the right direction. I'm quite new to this but am ready and very willing and keen to learn and expand my abilites.

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  • $\begingroup$ Note that GPC does not just separate proteins by Mr but by a combination of shape and Mr (though shape will be less important for you since most small proteins are quite globular) $\endgroup$
    – Nicolai
    Jun 19 '17 at 1:49
  • $\begingroup$ Thank you very much for this interesting piece of information. I will keep this in mind for a sample containing perhaps a mixture of globular and membrane proteins. $\endgroup$
    – Cello_101
    Jun 19 '17 at 10:31
  • $\begingroup$ I know this is an old question, but I would question why you don't just purify the lysozyme by crystallization from crude extract followed by polishing with IEC and SEC... $\endgroup$
    – Jeppe Nielsen
    Jul 7 '19 at 3:00
  • $\begingroup$ Here is source on how to crystallize directly from egg white. jbc.org/content/164/1/… $\endgroup$
    – Jeppe Nielsen
    Jul 7 '19 at 3:08
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Since lysozyme is so well studied, your best bet is to search the literature and see what methods other people use. See this paper, for example. They report that lysozyme can be affinity purified on Sephadex, but they also reference other methods in the introduction that you could take a look at.

Both of the methods you described sound reasonable. The only way to know which is best is to try them and see. In fact, the best approach, depending on your application, would likely be to use both methods for optimal purity. Even when I affinity purity a protein, I will still often perform ion-exchange as a polishing step.

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Here is what I have come up with. Thank you for all your help.

Ion Exchange Chromatography(IEC) will yield the purist product from a single application. The main reason is due to the fact that Gel Permeation Chromatography is compromised by the close proximity of Lysozyme (14.3 KDa) and Cystatin (12.0 KDA) in their molecular mass. Sephadax G100 separates a large range of molecular mass but the downside of large range is low sensitivity at such close proximity. It won’t be able to discriminate between these two proteins during the elution. Due to Cystatin only compromising 0.05% of the sample it will be even more difficult to pick up this contamination even though it is not large. Furthermore, Sephadax G100 will retain Lysozyme for very long as it is one of the smallest in the sample. Hence, a large amount will be retained and a diluted sample will be produced. It should also be taken into account that GPC has a low resolution, low capacity and does not permit high flow rates. GPC is the fastest and most simple method to produce a cruder sample in comparison to IEC. IEC in this case uses CM-Sephadex G25 which means no Lysozyme will be lost due to particle size properties and Mr allowing it to get caught in the matrix as opposed to GPC. This results in a more concentrated sample. If we compare the pI of all the proteins, Lysozyme (10.7) and Avidin (10.0) both are the only cations at initial pH 8.2 . This means lysozyme can easily be removed as pH is continuously decreased through the use of the cationic exchanger without denaturing it or any sample being lost. GPC is only useful as an additional step to IEC using G25 to further purify the sample through desalting and dialysis.

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  • $\begingroup$ Your answer is much better than mine. $\endgroup$
    – canadianer
    Jun 19 '17 at 15:25

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