I have a novel bacterial protein, which while isolating, doesn't come out in the supernatent as it forms inclusion bodies, thereby making it impossible to crystallize. Can anyone suggest what I should do in order to characterize it and see its localization in bacteria?
In silico characterization has been done, but it lacks significant homology.
You can decrease temperature to ~18°C and add osmoprotectants like betaine or sucrose. These worked for me.
In the medium during cell growth. From my thesis:
Overnight culture selected on appropriate antibiotics (BL21(DE3)pLyS with
pET16b: ampicilin 50 μg/ml and chloramphenicol 36 μg/ml; SG13009 with pQE30:
ampicilin 50 μg/ml and kanamycin 50 μg/ml) was grown in LB media supplemented with
1 M sorbitol and 2.5 mM betaine at 37°C, as described before (Blackwell and Horgan
1991) and with 100 mg/ml karbenicilin. When OD600 reached approx. 0.3-0.5 absorbance
units, IPTG was added to the final concentration of 0.5 mM to induce protein expression
and the incubation temperature was reduced to 20°C. Next day, the cells were collected
by centrifugation and resuspended in lysis buffer supplemented with protease inhibitors
(1 mM PMSF or 0.1 mM Pepstatin). This extract was loaded onto Ni-NTA spin columns
and spun at 2000×g. Columns were three times rinsed with washing buffer. Bound
proteins were washed out twice with elution buffer afterwards.
why dont you refold the protein by dissolving inclusion bodies into urea/ Gu-HCl and successive dialysis or dilution refolding? If your protein do not lose its activity, e.g. if protein can refold back correctly, you may get lots of protein from inclusion. On the other hand if yr protein is a membrane/ or membrane anchoring then you can truncate it to get soluble domain.
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