Efficient Expression of Bioactive Human Leptin in Escherichia coli in Soluble Fusion Form

Abstract

Leptin, a 16 kDa nonglycosylated hormone, is produced by mature adipocytes and functions primarily in the hypothalamus to reduce food intake and body weight. To explore a new approach for high-level expression of human Leptin in Escherichia coli, the human Leptin gene, synthesized according to the published sequence, was cloned into the vector pET32a to construct a fusion expression plasmid: Trx-Leptin/pET32a. Our data showed that more than 40% of the fusion protein Trx-Leptin was expressed in soluble form. After purified by Ni-IDA affinity chromatography, cleaved by enterokinase and applied Ni-IDA affinity chromatography again, purified Leptin with homogeneity over 96% was achieved. The biofunctional experiments of purified Leptin showed a significant reduction in food intake and body weight of female mice treated with Leptin by comparing with control mice, and it indicated that the purified Leptin has full biological activity. In addition, our expression system was a very lowcost and efficient prokaryotic expression system. So taken together, our results demonstrated that our expression system of bio-active Leptin provided a new method for producing Leptin in big scale and would be widely applied in commercial Leptin producing industries.

Engineered E. coli Nissle 1917 for delivery of bioactive IL-2 for cancer immunotherapy

“…IFNγ is an important cytokine for immunologically mediated antitumor responses. IFNγ can directly affect tumor cells by exerting antiproliferative and anti-angiogenic effects, as well as indirectly by increasing macrophage tumoricidal activity and attracting other immune cells 40 . We quantified IFNγ levels in the conditioned media from the co-culture of PBMCs and tumor spheroids.…”