E coli Lysis Buffer Recipe

Use this category for questions regarding problems manipulating proteins in molecular biology applications (expression, detection, etc.)

Moderators: mdfenko, leekaming

E coli Lysis Buffer Recipe

Postby Toxojulius » Nov 30 2007 6:40 pm

Hello,

I am attempting to express a recombinant protein in E. coli BL21(DE3) from a pET vector. I am searching for advice on making a good lysis buffer. I would like to stay away from sonication because it's time consuming, variable, and heats up my sample. The lysis buffer recipe I have now is the following:

10mM Tris-Cl pH 8.0
1mM EDTA pH 8.0
1mg/mL chicken egg white lysozyme (Sigma)
1% Triton X-100
5ug/mL DNase
5ug/mL RNase
1X Protease Inhibitor Cocktail (Sigma)

Here are my questions:
1. Should I incubate my cells first in the presence of lysozyme only (e.g. 30min on ice), then add the lysis buffer above but made without lysozyme? Is there an advantage of pre-treating cells in lysozyme before adding lysis buffer?

2. Usually I add the above lysis buffer above (with lysozyme) to my cells and incubate them shaking gently at 4C for 30min. then at room temperature for 30min. However, the suspension of cells never significantly clarifies. Is this normal? Are my cells still lysing?

3. When I centrifuge the lysate into soluble and insoluble fractions, the insoluble material forms a pellet that is almost as large as the original bacterial cell pellet. This had me thinking that I wasn't getting good lysis of my cells and I was just spinning whole cells down again. However, after I take off the supernatant and attempt to solubilize this insoluble pellet in 1X SDS Sample Buffer (w/5% beta-ME), the pellet is very, very difficult to solubilize. It takes numerous heating-then-vortexing cycles to get the pellet to go into solution into SDS Sample Buffer. Is this normal? Why is the insoluble pellet so hard to solubilize in SDS? Is there something in my lysis buffer that is causing the pellet to be really difficult to dissolve in SDS Sample Buffer?

4. Does anybody have any suggestion for a good lysis buffer recipe? If so, could you tell me what is in it and why?

Thanks!
Toxojulius
technician-in-training
technician-in-training
 
Posts: 11
Joined: Mar 09 2007 2:01 pm
Location: Vermont

Postby jk » Dec 01 2007 7:51 am

You don't really need triton in your lysis buffer.

I have the best lysis when I suspend my cells in 5mL lysis buffer (20 mM Tris, 300 mM NaCl, pH 8.0, protease inhibitors) per 1 g cell paste. then I add lysozyme to 1 mg/ml, and DNase. DNase needs Mg2+ for activity, so you should add 0.1 mM - should be enough. Incubate 30-40 min on ice.
It is probably the DNA which makes your pellet difficult to solubilize.

if you want to lyse the cells very efficiently you can do it with BugBuster (Novagen) or similar products.

cheers
jk
jk
supertech
supertech
 
Posts: 63
Joined: Jul 18 2003 7:04 am

Postby Toxojulius » Dec 01 2007 1:33 pm

Thanks for the post. I would like to stay away from proprietary lysis buffers since nobody is allowed to know the exact composition, even though they work well.

I am confused why you don't have ANY detergent in your lysis buffer? I always operated under the assumption that lysozyme needed access to the cell wall. I thought that no access would occur unless there was some detergent to remove the outer membrane lipids of E. coli.

I thought Triton-X would an appropriate detergent to solubilize the cytoplasmic membrane, but I included EDTA in my lysis buffer to chelate the divalent cation crosslinks stabilizing the outer membrane, which should allow access for the lysozyme to attack the peptidoglycan layer. This seemed reasonable to me, but again, the resuspended cells never significantly clarify after incubation in this lysis buffer. I always assumed lysis would be indicated by a decrease in turbidity and an increase viscosity due to released nucleic acid.

I would appreciate some feedback on the composition of my lysis buffer and any advice on lysis of E. coli expressing recombinant protein without the use of sonication.

Thanks!
Toxojulius
technician-in-training
technician-in-training
 
Posts: 11
Joined: Mar 09 2007 2:01 pm
Location: Vermont

Postby relaxin » Dec 03 2007 9:37 am

I think EDTA and RNase are not needed.

increase TrisHCl to 50 mM
add 200 mM NaCl
add 1 mM DTT
reduce Triton X-100 to 0.5%

1. Resuspend cell paste at 1 ml lysis buffer/g cells.
2. Add lysozyme at 300 ug/ml. Incubate at 4 C for 1 hr.
3. Add MgCl2 tp 10 mM and treat with DNase at 4 C for 30 min.
Not affiliated with any company. Mention of a specifc product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 6633
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Postby celllysis » Dec 05 2007 11:10 pm

Here is a recipe that I use. It includes a recombinant human lysozyme called Lysobac. I have found that the recombinant is more active and consistent than chicken.

10 mM Tris.Cl (pH8.0)
100 mM NaCl
1 mM EDTA (pH8.0)
5% Triton X-100
Lysobac(1 mg/ml)
celllysis
newcomer
newcomer
 
Posts: 1
Joined: Dec 04 2007 12:20 pm

Postby RRam » Dec 06 2007 11:00 am

Toxojulius wrote:Thanks for the post. I would like to stay away from proprietary lysis buffers since nobody is allowed to know the exact composition, even though they work well.

I am confused why you don't have ANY detergent in your lysis buffer? I always operated under the assumption that lysozyme needed access to the cell wall. I thought that no access would occur unless there was some detergent to remove the outer membrane lipids of E. coli.

I thought Triton-X would an appropriate detergent to solubilize the cytoplasmic membrane, but I included EDTA in my lysis buffer to chelate the divalent cation crosslinks stabilizing the outer membrane, which should allow access for the lysozyme to attack the peptidoglycan layer. This seemed reasonable to me, but again, the resuspended cells never significantly clarify after incubation in this lysis buffer. I always assumed lysis would be indicated by a decrease in turbidity and an increase viscosity due to released nucleic acid.

I would appreciate some feedback on the composition of my lysis buffer and any advice on lysis of E. coli expressing recombinant protein without the use of sonication.

Thanks!


Your idea of adding the detergent is elite. Lysozyme works differently with the age of the cells too. Moreover it also adds up as an impurity when you want to extract the soluble protein.
To me when the lysis has occured it takes little bit extra time to clarify the sample. U may check the protein content before and after the lysis along with the lysis buffer as a blank for the after lysis sample.
I have used glass beads with shaking which i got comparable results with sonication. Heat liberation also very less. If you are interested do let me know . I can give the protocol.
RRam
supertech
supertech
 
Posts: 68
Joined: Jun 20 2007 9:25 am

Postby himuthu » Dec 06 2007 12:50 pm

Hi,
When I use lysozyme to break my e.coli, I used to sonicate brifely (just few pulses) to break the DNA. This helps to clarfy the lysate as well as facilitate running a purification column later.

If you don`t want to use sonication, you can also try other detergent based lysing solutions like BPER from Pierce.
Muthu Meiyappan
Xtal BioStructures Inc.
http://www.xtalbiostructures.com
himuthu
supertech
supertech
 
Posts: 61
Joined: Feb 13 2003 12:14 pm
Location: Watertown, MA

Re: E coli Lysis Buffer Recipe

Postby Duke » Dec 13 2007 6:45 am

Toxojulius wrote:Hello,

I am attempting to express a recombinant protein in E. coli BL21(DE3) from a pET vector. I am searching for advice on making a good lysis buffer. I would like to stay away from sonication because it's time consuming, variable, and heats up my sample. The lysis buffer recipe I have now is the following:

10mM Tris-Cl pH 8.0
1mM EDTA pH 8.0
1mg/mL chicken egg white lysozyme (Sigma)
1% Triton X-100
5ug/mL DNase
5ug/mL RNase
1X Protease Inhibitor Cocktail (Sigma)


The recipie sounds good to me despite you do not need Triton (it is also difficut to get rid of it in subsequent purification steps). RNase is also not necessary as RNA does not influence the viscosity of the solution.
It is pretty hypotonic which should help breaking the cells at lysozym digestion.
DNase needs Mg (1mM) and Ca (0.1mM) in oder to funcion. If you use Mn instead of Mg, DNase will generate somthing like "blunt end" breaks.

But be aware, EDTA (and eventually EGTA in the Protease Inhibitor cocktail) will lower the concentrations of Mg and Ca!! That is the function of EDTA/EGTA as protease inhibitor. It scavanges the heavy ions which are used by some proteases as co-factors.

Here are my questions:
1. Should I incubate my cells first in the presence of lysozyme only (e.g. 30min on ice), then add the lysis buffer above but made without lysozyme? Is there an advantage of pre-treating cells in lysozyme before adding lysis buffer?


I do not see any need to pretreat the cells with lysozym as long as your lysis buffer is hypotonic.

2. Usually I add the above lysis buffer above (with lysozyme) to my cells and incubate them shaking gently at 4C for 30min. then at room temperature for 30min. However, the suspension of cells never significantly clarifies. Is this normal? Are my cells still lysing?


Your cells are not dissolved but lysed! That means the insoluble fraction does not dissapear but the soluble cytosol is just released. Therefore a cell SUSPENSION (not SOLUTION!) is never clear, esp not, when the cells are resuspended in small volumes (like 5 ml per 1 g).
That also explains your observation that the pellet does not get smaller afer lysis. The portion of insoluble parts ist still the same. ;-)

3. When I centrifuge the lysate into soluble and insoluble fractions, the insoluble material forms a pellet that is almost as large as the original bacterial cell pellet. This had me thinking that I wasn't getting good lysis of my cells and I was just spinning whole cells down again. However, after I take off the supernatant and attempt to solubilize this insoluble pellet in 1X SDS Sample Buffer (w/5% beta-ME), the pellet is very, very difficult to solubilize. It takes numerous heating-then-vortexing cycles to get the pellet to go into solution into SDS Sample Buffer. Is this normal? Why is the insoluble pellet so hard to solubilize in SDS? Is there something in my lysis buffer that is causing the pellet to be really difficult to dissolve in SDS Sample Buffer?


The solubility depends on the concentration (volume of sample buffer). In the pellet there are many different ingredients like lipids, insoluble cells wall parts, organells etc. which are just not soluble. with the SDS buffer you usually try to solubilize insoluble proteins of the pellet, but there are losts of other contents.
For proteins a heating-vortexing-cycle should be fine. If you have to many unwanted contents in the sample, you might just end up with a smear on the gel....


4. Does anybody have any suggestion for a good lysis buffer recipe? If so, could you tell me what is in it and why?

Thanks!


Your recipie without Triton and DNase sounds good to me. (Plus Mg/Ca)

You might add a brief sonification step at the end which gains a lot esp. in bacterial lysis.

Edit: There was a mistake in my reply: I meant DNase not RNase is good in your buffer... sorry...
Last edited by Duke on Dec 15 2007 3:44 am, edited 1 time in total.
Duke
technophile
technophile
 
Posts: 36
Joined: Oct 03 2007 7:36 am

Postby Toxojulius » Dec 14 2007 10:59 am

Thanks for your post Duke. Your post was really helpful. I have some more questions.

1. I keep seeing Benzonase as a component of some E. coli lysis buffers. I haven't had any experience using Benzonase, but I assume it's just DNase. Has anybody used this stuff?

2. The protein I am expressing in E. coli is His-tagged. I am worried about putting EDTA in my lysis buffer because when I apply the lysate to the column, I don't want the immobilized metal on the column to be chelated by the EDTA. However, it seems like many lysis buffers include EDTA to destabilize the divalent crosslinking in the outer membrane of E. coli. Is there a critical amount of EDTA that I can get away with adding to the cells to facilitate lysis, but that won't affect binding of the IMAC column?

3. Why is it good to have a hypotonic solution when using lysozyme?

4. If I sonicate my cells after lysozyme treatment, do I need to add DNase at all? Will sonication break up the DNA sufficiently to prevent viscosity?

Thanks!
Toxojulius
technician-in-training
technician-in-training
 
Posts: 11
Joined: Mar 09 2007 2:01 pm
Location: Vermont

Postby relaxin » Dec 14 2007 11:41 am

1. Benzonase is a genetically engineered endonuclease that will degrade DNA and RNA. Regular DNase will do the job. For more detailed info, please click on the following link:
http://www.merck.ru/servlet/PB/show/170 ... zonase.pdf

2. EDTA also affects the DNase digestion. You can omit it.

3. Hypotonic solution is used to aid "bursting" of the cells after the cell wall is weakened by lysozyme.

4. If you use sonicate, no need to use DNase. They serve the same purpose.
Not affiliated with any company. Mention of a specifc product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 6633
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea


Return to Protein Methods

Who is online

Users browsing this forum: Google [Bot] and 5 guests