Bacterial Periplasmic protein localization - B-lactamase- alkaline phosp Fusion experiments (Feb/08/2005 )

Dear colleagues

I have been desperately looking for a B-lactamase- alkaline phosphatase fusion systems or vectors. I have a bacterial protein that was predicted to be periplasmic and would like to prove it experimentally.

Have anyone done similar experiments that he or she can share?

we checked the periplasmic location of one of our proteins by mild periplasmic lysis of the bacterium, followed by a westernblot. antibodies to an exclusively cytoplamic protein served as control for the differential lysis.

mike

Simple way to prove the periplasmic localization of the protein is to check for presence of a signal peptide in the sequence. Further, osmotic shock can be employed to selectively extract periplasmic proteins (yield will be lower). suspend the cells in 205 sucrose in the buffer of your choice along with 10mM EDTA, incubate for 30 min, pellet out the cells by centrifugation and resuspend in icecold water for 30min and collect the supernatant.

expression as a periplasm localising fusion protein does not prove the point because you have artifially induced the localization with an appended signal.

Sharath.B
Research Fellow
National Chemical Laboratory
India

Thank you very much for sharing your opinions, that was very helpful.

MolecMicrobiol

I am also trying to extract periplasmic protein now~
My protocol is similar with above.

Fractionation buffer(10mL):
2g sucrose
Tris-HCL(pH8.0)10mM
Na2EDTA 5mM
(conc. of Tris-HCl and Na2EDTA can be adjusted.)

1.Resuspense 50mL cell pellet with 0.4mL fractionation buffer, waver at RT for 10min.
2.Centrifuge at low temperture,12000rpm,10min,throw supernatant away
3.Add 0.4mL MgSo4 5mM, votex 10 min to mix
4.Centrifuge at low temperture,12000rpm,10min,periplamic protein is in the supernatant.



Good Luck!