expression of MBP fusion protein - leaky and very low yield (Aug/02/2010 )

This is a modified pMal vector. We put the MBP protein in the C terminus, rather than the N terminus.

I first tried to express in BL21(DE3)STAR (as always). induced with IPTG (tried 0.3mM, 0.5mM, 1mM) when OD600 reached 0.5-0.6; expressed in 37C (2-4h), 25C,18C,14C (Overnight),
I took out samples before induction and after expression, measured the OD (to make sure I load the similar numbers of cells to the SDS-PAGE). Boiled in sample loading buffer , then run the gel.

I didn't see any difference between the un-induced and the induced.

I tried to purify some proteins, and I did get the band I wanted.

if you got your protein then it was autoinduced.

Thanks!

The yield was extremely low, and I need a lot of proteins to do crystallization..... Oh my!
I was wordering if there's a specific cell strain I need to try....

mdfenko on Tue Aug 3 14:51:24 2010 said: