I am trying to lysate BL21 DE3 E. coli for the purification of recombinant proteins Blo t 2 (14.5KDa). I couldnt achieve results in denaturing condition but in native conditions, when using lysozyme (also 14.5KDa), I found my protein band in SDS PAGE gel. Since this may be lysozyme and not the protein at all, I used sonication and freeze/thaw cycles for cell lysis instead and again achieved no results.
lysis buffer containing 10mM imidazol, 500mM NaCl, 20mM Tris-HCl (pH=7,5),
What protocol do you recommend?
It depends on your set-up, but generally, you will do it as follows:
1) Harvest your E-coli cells using centrifugation
2) Resuspend your cells in lysis buffer (Imidazole at this stage is generally not necessary, but depending on your protein, you may want to add some glycerol. Generally, something like 300mM NaCl, 50mM Tris pH 7.5, 5% Glycerol, and some DNAse1 and protease inhibitor cocktail (cOmplete EDTA-free) should work)
3) Sonicate (or French press) your cells. I personally use a Q500 sonicator with a standard half inch probe, 30% intensity, 3s on / 3s off, for 3 minutes of sonication time, or 6 minutes total time.
4) Clarify your lysate using high-speed centrifugation - 10.000g or more for at least 20 minutes at 4 degrees celsius is advised.
5) Filter your lysate, first through 0.8uM, then through 0.45uM, then through 0.22uM syringe filters
6) Now at this point you can perform pulldowns on small quantities of your lysate, and take a small sample of your pellet. This will tell you whether your protein is expressed, and whether it remains soluble in the lysate or whether it is in the pellet and you need to troubleshoot your construct design or purification strategy.
E. coli are not so rigid that you need to use extremely complicated protocols using freeze/thaw cycles that may result in issues with your protein. Thorough sonication on ice in the presence of protease inhibitor is generally sufficient. You should always check whether you can detect your protein in the pellet after clarifying the lysate, because that tells you whether the protein is insoluble, or whether you just did not have sufficient expression of your protein of interest.
Completely agree with what Michael Adams said so try their suggestions first. If they don't work and you're sure that your protein is actually expressing at detectable levels, then E. coli can be lysed with a (comparatively) "gentle" method just in case the sonication or freeze/thaw is causing issues for your protein. This method causes minimal protein damage but may have lower yield:
For small amounts of cells, you can use a detergent-based lysis buffer.
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I agree with the two previous comments. It is essential that you check whether the target protein is soluble or whether it is insoluble and "hiding" in the pellet containing cell debris. Whatever the method, mechanical or chemical lysis, you can always check its efficiency by measuring the OD600 before and after the procedure.
Nishelle Dsouza
Hi everyone
as Pierre Béguin and Michael Adams said, It is very important to know if your protein is soluble in supernatant or Inclusion body in the pellet, and I guess :
if you have overexpressed protein, it is in your bacterial pellet.
first, did you see the 10kd band of the protein ladder in your SDS-page gel?
second, what is your vector for protein expression, and what are the conditions for expression like temperature and concentration of IPTG?
third, what is your strain for protein expression?
I attached a picture that the difference is just the strains for protein expression.
Hello Nishelle, it is possible that high salt content could affect your specific protein migration. If you do not want to dialyze the lysate to reduce the salt concentration, then you can start off with only 50mM Tris, 1mM PMSF, pH 7.4 as lysis buffer in your sonication process. I have observed dissimilar migration of my protein in high salt buffers.
you may omit lysozyme and adopt physical methods of cell disruption, such as Homogenization and sonication. This will rule out the possibility of lysozyme detection along with your expressed protein. As mentioned in previous comments, you should first check the expression of your protein, if it is soluble or insoluble. For this, after lysis run small quantity (May be 1 or 2 uL) of your cell debris on SDS PAGE along with other soluble fractions.
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