try experimenting with different, especially protease-deficient, E coli strains to express the protein and try different methods to lyse the bacteria (sonication, french-press, emulsification, bead-beater, mortar & pestle under liquid nitrogen).
on the other hand, if you are lucky, you are just proteolysing some surface loops and can still purify and crystallise the protein. This was done on purpose for the cap-binding complex, see: Crystal structure of the human nuclear cap binding complex. Mazza C, Ohno M, Segref A, Mattaj IW, Cusack S. Mol Cell. 2001 Aug;8(2):383-96. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 15 Feb 2012, at 14:09, Sivasankar Putta wrote: > Dear All, > > Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi > domain) DNA binding protein, that we are expressing at 18 degree Centigrade > in E. Coli. The protein appears to degrade during purification; we have > protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM > EDTA and 1mM DTT throughout during purification ( right from lysis stage). > We handle the protein at 4 degree Centigrade. > > Can you please suggest what precautions we can try to avoid such degradation > ? > > Please find the attached gel picture regarding protein > > Sivasankar Putta > > > > <proteingel.pdf>
