Alpha-Amylase expression and purification - (Feb/05/2008 )

Hi;
I have cloned B.licheniformis alpha-amylase to S.cerevisiae and confirmed the expression of active enzyme on starch agar plates.
However; I cannot get the yeast to express the protein in liquid medium even if they grew to OD= 7.
I used 20mM CaCl2 to stabilize the amylase but still I cannot see the protein band in PAGE.
Any suggestions??
Thanks.

-Tunc-

Which polymerase did u use to amplify your onterested gene?Did you checked its sequence? Are u using right secretion signal? It may be mutated.you shouldcheck the sequences try to cloen it again.

-mhmtcn-

QUOTE (mhmtcn @ Mar 18 2008, 11:44 PM)

Which polymerase did u use to amplify your onterested gene?Did you checked its sequence? Are u using right secretion signal? It may be mutated.you shouldcheck the sequences try to cloen it again.

Yes, the actual problem turned out to be at the secretion signal. The degradation on starch plates were because of the accumulating enzyme inside the cells. That is why we could not see any in liquid media. But thanks anyway for your concern. WE are recloning the enzyme now.

-Tunc-

Be careful when u are amplifiying the gene. You should use a polymerase which has strong proof reading activity. You may not concentrate on your expeirment well last time. So this time do it properly and make triplicates. You should also listen your partner/s. They may think something that you missed. Anyway take care of yourself.

-mhmtcn-