I have inserted laccase gene in pET32 vector, verified the sequence, cloning site and transformed it into E.coli BL21(DE3) as well as E coli BL21(DE3) pLysS. I have also tried using carbenicillin antibiotic and lower IPTG concnetration (0.4mM) at 20, 25 and 37 degree C for various incubation times from 3 Hr up to 18 Hr. But in every case I am seeing the target protein band of around 100 KDa irrespective of IPTG induction. Has anyone experienced such problems in pET32 Xa/LIC mediated cloning? Thanks