Heterologous protein extraction in yeast for enzymatic activity test - (Mar/24/2011 )

Hi,

I've been trying to extract protein from yeast using sample buffer (0.06M Tris-HCl pH6.8, 5% glycerol, 2% SDS, 5% 2-mercaptoethanol)and heat for 3 min at 95 degrees. I've managed to do a buffer exchange and his-tagged purification to get my desired protein band with some background proteins. My protein is his-tagged, expressed with Gal1 promoter.

Would the sample buffer denature my protein (an enzyme) and hence affect its activity? Which protocol should I follow if I want to extract my protein in the active form?

Also, despite expressing it for a long time (24, 48h), there is no obvious over-expression of my protein from sds-page analysis. Is this normal for yeast expression system?

Regards,
simlez

simlez on Fri Mar 25 02:56:43 2011 said:

Hola sample buffer is suitable for denaturing and analyze denatured proteins in PAGE. to see amount of each, but to isolate active forms you have work in "natural" conditions to avoid lost of estructure and activity. So you have to broke your cells of any of the physic method(balls agitation?) avoinding an excess of temperature separate the particulate fraction and run your specific tag purification. About inductions I believe that to metabolize galactose and induce the expression glucose has to be consumed. how has changed OD in the induction period. Do you work in baffled flask or in fermenter ? do you have alcohol formation?. Buena suerte

protolder on Fri Mar 25 07:26:15 2011 said:

Mrs Shep on Thu Apr 14 12:17:21 2011 said:

Thanks for your suggestion. I have a question - after growing for 4-5 days, what would the growth phase of your culture be? I'm afraid of growing the cultures to stationary phase as they tend to form thicker cell wall which is harder to lyse.

protolder on Fri Mar 25 07:26:15 2011 said:

Thanks for your suggestion. My protein could be a membrane protein, still working to extract it under non-denaturing condition.
I work with baffled flask. Does alcohol formation affect protein expression?

hola, I´m not sure, if yeast continue growing probably not but if you reach a growth plateau for alcohol excess I don´t know if in this phase could protein synthesis be done but I think all these process are repressed. If I were you I´ll add sugar each day, after ethanol produced of the anterior addition has been metabolized for grow. if grown is stopped in presence of carbon source think in addition of nitrogen source, and the culture will continue growing, if no, could be necessary any salts or vitamins.Buena suerte