PERIPLASMIC PROTEIN ISOLATION.

[Cornelius Krasel]

nitika at imtech.ernet.in wrote:

: I am isolating the periplasmic fraction of E.coli for release
: of an enzyme. Am presently usind osmotic shock treatment( sucrose shock)
: as well as combinee treatment with lysozyme. Both the methods are leading
: to the lysis of the cells without miantenance of intact spheroplasts.What
: could be the reason? Kindly suggest another method. Thank you.

Two years ago I used the following method for the release of antibody
Vl fragment expressed in E. coli periplasm:

- Add 2 ml 1 mM EDTA in BBS (which is 160 mM NaCl, 200 mM boric acid,
  pH 8 with NaOH) per gram wet cell weight.
- Stir the solution at least 30 minutes at 4 degrees Celcius.
- Get rid of the spheroblasts by centrifugation (SS34, 20 min, 20000 rpm,
  4C). 
- Get rid of membrane fragments etc. by sterilfiltration of the supernatant.

Hope it helps, Cornelius.

--
/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */
/* email: krasel at alf.biochem.mpg.de, krasel at vms.biochem.mpg.de            */
/* "People are DNA's way of making more DNA." (R. Dawkins/anonymous)      */