Trouble producing GST fusion proteins in BL21 Star E.coli
I'm trying to produce GST fusion proteins to use in pull down experiments. The genes are successfully cloned into pGEX-6P-1 plasmids and confirmed by sequencing and transformed into BL21 Star E.coli for expression. I cannot, however, seem to produce protein by induction with IPTG. I have tried a range of IPTG concentrations up to 2mM and induced at 28oC, which was recommended to me. When I Western Blot this using anti-GST antibody I do get bands, 1 at ~26 KDa which is roughly he size that would be expected for the GST on it's own (there shouldn't be any) which increases in intensity with time but not IPTG concentration. There is a strong band at ~50 KDa which I have no idea what it could be and sometimes I get quite a faint band above this of ~60KDa. The proteins I want to tag are between 54 and 60 KDa so it's probably not these.
I was hoping someone might have had similar problems or may have any general advise for me to produce the proteins that I want.