I have a problem with expression of untagged fragments of proteins in pet28a vector.
I try do do in vitro
expression in E.coli
. When I put lysate after sonication & centrifugation on the gel and stain it with Coomassie, I can see nicely expressed protein bands but the size of proteins is wrong (and - what is even more interesting - the same for all fragments). My fragments are N- and C-terminal fragments of the same protein.
I sent bands to Mass spectroscopy but peptides identified in samples don't match my protein.
I'm sure I didn't make mistake during transformation (repeated) and contructs are sequnced (so there is no premature STOP codon etc)
I want to mention that when I tried to do expression of the same fragments but their version with HIS-SUMO tag from similar vector everything was ok.
I would appreciate help very much