Enhanced production and refolding of human leptin expressed in Escherichia coli

The present study describes enhanced production and simplified refolding of a pharmaceutically important protein, leptin, expressed as inclusion bodies in a bacterial expression system. The gene encoding leptin was amplified by RT-PCR methodology, cloned in pTZ57R/T vector by employing dA.dT cloning strategy and then subcloned in T7lac promoter-based pET-22b (+) vector to generate pET-LP expression plasmid. E. coli strain BL21 (DE3) CodonPlus transformed with pET-LP produced a prominent protein of ~16 kDa upon induction with IPTG/lactose, which represented >30 % of the total E. coli cellular proteins. A study of the effect of medium composition on expression level and cell densities revealed that higher cell densities equivalent to ~20 g/L may be achieved in YNG autoinducing medium without compromising the expression level. Whether induced with IPTG or lactose, the target protein expression was in the form of biologically inactive inclusion bodies (IBs), which could be solubilized using a mild detergent i.e., N-lauryl sarcosine (NLS). Refolding was achieved by dilution in the presence of cysteine and cystine followed by stepwise removal of NLS by employing dialysis. The strategy used in the study for refolding is simple, straight-forward and may successfully be employed for proper folding of those recombinant proteins that are expressed as IBs and contain just one or two disulfide bonds.