I would like to take some advice on a better approach for my protein extraction, which is a hydrophobin protein. I use BL21 cells for my expression culture and sonication to obtain the cell-free extract, which is not giving efficient results. Previously, for the culture of flavoproteins, I have used lysozyme with PMSF together with sonication a couple of times. In this method, I incubated the cells in buffer at room temperature before sonication, which fortunately gave me good results.
I wanted to ask if this approach really helps and how exactly it affects the process. Or does it just depend on the buffer and sonication cycle used for the lysis solution?
Please help with some of your suggestions and clarification on this topic.
Hi Vaibhav,
Using lysozyme and PMSF during sonication is a good approach. If you can see that your pellet of broken cells is much smaller than your original wet pellet that means your cell lysis is good. And if you don't have a lot of fragmentation of your protein on the gel that means more protease inhibitors (such as EDTA or inhibitor cocktail) won't help either.
Since you know your protein is hydrophobic the most likely scenario is that it isn't folding well in the first place. I suggest running some of the insoluble cell pellet on SDS-PAGE gel together with the soluble supernatant. If there is a big band corresponding to your protein then it is in fact being made and you still have a chance. If it is only in the insoluble pellet then you can try adding detergent such as Triton X-100 to your lysis buffer to bring some of your protein up into the soluble fraction. There are also refolding methods which you can try, but I'm not familiar with those. Good luck!
Hi Vaibhav
I agree with J. Wagner. I would recommend using different expression strains, since some express the protein better than others, BL21, Rossetta, Sheuffle, etc. Likewise, determine the appropriate harvest time, since over time, protein insolubility can increase. In addition. To extract your protein you can use different methods such as detergent lysis, freezing-thawing and sonication, each protein is different.
Greetings and good luck.
A hydrophobic protein (a rather vague description) will most likely associate with the membranes which can be pelleted after lysis. As Jonathan mentioned, you may try to add detergent to your lysis buffer or treat the pellet with it. I suggest evaluating the soluble/insoluble ratio at different pH across the spectrum, as hydrophobic proteins are more prone to isoelectric aggregation effects. If your protein doesn't have to be intact, you can extract it from the post-lysis pellet in urea or guanidinium chloride buffer. If you require it to be soluble and potentially still intact and buffer pH doesn't fix it, I suggest adding a large soluble fusion partner like MBP or GST as baloon which may be cleaved off at a later stage if required.
As it comes to cell lysis, lysozyme will be more gentle to your extract than sole sonication, though the lysozyme is an added impurity (it tends to aggegate into the lysis pellet). When doing without, you should add up to 20mM EDTA to your lysis buffer to destabilize the cell walls and sonicate thoroughly and on ice to reduce thermal denaturation.
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