Always expressing truncated protein
Posted 12 December 2010 - 01:48 AM
So I have this GST-tagged protein vector with Amp resistant and IPTG induction.
I transformed the cell in Rosetta(DE3)pLysS, plate in LB + amp, grew in TB media + amp + 0.2% glucose, until certain OD600 0.7 or 1, then depending on the temp I grow (37C or 16C) I induce with IPTG for certain time, and lyse the cell etc.
The problem is that the protein is always truncated. It's been truncated in the cell itself. I've tried inducing at 16C 16hours or 37C 2 hours, with LB or TB, with different IPTG concentration but they always make truncated protein. I know that the protein is the truncation of GST-protein I wanted because it binds to GST column. I also know that it happen in the cell (not lysis buffers) because I lysed, run at gel then comparing with the uninduced and the truncated protein is there.
I mean, in all my protein prep I've always get either no expression or huge expression of the correct protein. But a truncated protein with 0 correct protein has never happened.
I also have sequenced the plasmid and it's all good. No insertion/deletion, no frameshift, no duplication, nothing. Please enlighten me on what might have happened?
FYI the protein is supposed to be 65 kDa and it's always, always make 57 kDa protein. I compared this to ladder and to another protein to make sure. I also use fresh DTT and loading dye each time.
Posted 12 December 2010 - 02:05 PM
Posted 13 December 2010 - 09:59 AM
Posted 13 December 2010 - 02:42 PM