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 bio-functional 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 low-cost 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.

Keywords: Affinity purification; Enterokinase; Escherichia coli; Fusion expression; Human leptin; Trx.

Fig. 1

Schematic representation of the expression vector pET32a–rhLeptin. rhLeptin was expressed as a fusion protein with the Trx. To facilitate peptide cleavage from the fusion protein, an engineered EK site, DDDDK (indicated by a vertical arrow), is created between the His-tag and rhLeptin

Fig. 2

Expression optimization of Trx–rhLeptin. a Expression of Trx–rhLeptin at 37, 30 and 25°C. Lane M: molecular weight marker; lane 1: non-induced bacteria lysate; lane 2–4 represented bacteria lysate from cell with 0.1 mM IPTG induction at 37, 30 and 25°C. b SDS-PAGE analyses of target protein expression under different temperatures. Molecular weight marker was shown in the left lane; lane 1: non-induced bacteria lysate; lanes 2–4 are soluble proteins when cultured at 37, 30 and 25°C, respectively; lanes 5–7 are the corresponding insoluble proteins. The arrow indicated the location of the recombinant Trx–rhLeptin fusion protein

Fig. 3

Purification of Trx–rhLeptin. a Ni-IDA affinity chromatography of Trx–rhLeptin by using LPDataView (BIORAD). b The eluted fusion protein showed about 95% or more purity by electrophoretic analysis with 15% SDS-PAGE as analyzed by the Bandscan software (BioMarin Pharmaceutical Inc, UK). Lane M: molecular weight marker; lane 1: supernatant of cell lysate; lane 2: protein fractions eluted from the immobilized metal affinity chromatography

Fig. 4

SDS-PAGE analyses for the rhLeptin preparation. a Trx–rhLeptin cleaved by 1, 2, 3 U EK at 30°C for 12 h (12% SDS-PAGE). Molecular weight marker was shown in the left lane; lane 1–4: protein mixture lysed by 0, 1, 2, 3 U EK. b rhLeptin purified by Ni-IDA affinity chromatography (15% SDS-PAGE). Lane M: molecular weight marker; lane 1–2: SDS-PAGE of rhLeptin in the absence and presence of DTT

Fig. 5

Intraperitoneal administration of rhLeptin into normal female mice. a, b Graphs showing the average change in food intake or weight gain in percentage over the weight at day 0 of normal female mice injected either with rhLeptin or vehicle control solution. c, d Graphs showing Cumulative food intake or weight gain (in mg/day) for the period of treatment and thereafter. Each value presents mean ± SD of three independent experiments. * P < 0.05 as compared with group in the presence of vehicle control solution only