Dear all,
I add sucrose to my lysis buffer (E. coli culture) in order to prevent aggregation of my target protein during lysis. When I centrifuge the lysis mixture (13000 g for 15 minutes), I get my protein in the supernatant (at least seemingly, checked by SDS-Page).
I now suspect that the sucrose solution, due to its high density (I use 1 M sucrose) hinders the inclusion bodies from settling down during centrifugation and that I therefore rather detect floating inclusion bodies, and not truly soluble protein.
Can anyone confirm this effect? What can I do to circumvent this, if it poses a problem? If not, I'm happy, since then I would really have soluble protein :)
Thanks for all answers,
regards,
Peter
1 M sucrose is 342 g/l, or 34.2% (w/v). This concentration is in the range used for preparative sucrose gradient ultracentrifugation to purify subcellular fractions. I'm pretty sure that true inclusion bodies will pellet in this concentration in an ultracentrifuge run, given sufficient g-force and time. However, if you are using a regular centrifuge, the g-force and time may be insufficient to pellet the inclusion bodies through 1 M sucrose. You could test this by using an ultracentrifuge at 100,000 x g and an overnight spin.
Incidentally, proteins that are overexpressed in an insoluble form are not always in inclusion bodies. They may just be precipitated.
Inclusion body density is usually > 1.3g/mL so they pellet with very mild centrifugation. They can have 50-80% void volume and thus increase in density with their suspending fluid, I'd spin your sucrose solutions at higher g force for a longer time, like Adam suggests.
I don't understand why use sucrose to circumvent aggregation as your protein is already in inclusion bodies? You would simply get the inclusion bodies by centrifugation at high rates in a buffer, wash them by another centrifugation as you have done but without sucrose.
We classically did that to purify the inclusion bodies. And then try to obtain the protein soluble by using low % of detergent such as NLS (0.3% final concentration in you buffer) for example and lyse it by gentle manual potter. Let it 1H at room temperature and then centrifugate it at 15000g &10 min at 4°C. And after, make gentle dialysis. And after purification on exclusion chromatography which very efficient to subctract the detergent.
All the best
For inclusion body/precipitated proteins the protein is already aggregated as Elisabeth points out and is usually mis-folded, but there are exceptions. Does microscopic examination show grey or refractile inclusion bodies where the grey ones are usually associated with lipids while the refractile ones are very dense? If you are using sucrose to help purify the inclusion bodies then the process can be done differently or more simply as Elisabeth points out but be aware that the inclusion body density can change with solvent conditions as Edward mentions.
To clear up the issue, I want to separate solubilized protein from remianing inclusion bodies by this approach. I am not interested in the IBs, I just need a way to get rid of them as contaminant.
What is the scale of your process and do you have access to dynamic light scattering (DLS)? If your scale is in hundreds of milliliters then ultracentrifugation may not be convenient otherwise Adam's point is applicable. Is the lysis method by homogenization or lysozyme/hypertonic solutions? After your first 13 K x g spin a DLS may help to assess particulate distribution as well as aggregate sizes in the supernatant. Is it possible to use macro, micro, or ultrafiltration methods in a TFF mode? Is it possible to substitute low concentration mixtures of arginine and glycerol for the sucrose to avoid the high density but yet stabilize the protein?
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