Efficient Secretion of Biologically Active Recombinant OB Protein (Leptin) inEscherichia coli,Purification from the Periplasm and Characterization

Guisez, Yves; Faché, I; Campfield, L. Arthur; Fj, Smith; Farid, Alyaa; Plaetinck, Geert; Heyden, José Van der; Tavernier, Jan; Fiers, Walter; Burn, Paul; Devos, René

doi:10.1006/prep.1997.0836

Cited by 33 publications

(20 citation statements)

References 36 publications

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“…The pLPPsOMPAhOB plasmid harboring the S120A/T121A mutation was electroporated into Escherichia coli MC1061 cells. This allows the expression of untagged, properly folded S120A/T121A human leptin in the periplasm of E. coli MC1061 cells (28). The periplasmic fraction was prepared by an osmotic shock procedure (28), and complete protease inhibitor (Roche Applied Science) was added.…”

Section: Structural Superposition and Molecular Modeling Of Mouse Lepmentioning

confidence: 99%

Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

Peelman

Beneden

Zabeau

et al. 2004

Journal of Biological Chemistry

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131

169

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The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.

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Section: Structural Superposition and Molecular Modeling Of Mouse Lepmentioning

confidence: 99%

Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

Peelman

Beneden

Zabeau

et al. 2004

Journal of Biological Chemistry

Self Cite

131

169

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“…Up to date, however, secreted production of leptin in E. coli was rather inefficient. Guisez et al [13] used the OmpA signal peptide for the secretion of human leptin which was encoded by the modified sequence based on the E. coli preferable codon usage. The amount of leptin secreted into the periplasm was 2-4 mg/ml/A600 nm cells, and only 2 mg of human leptin could be purified from a 3-l culture.…”

Section: Discussionmentioning

confidence: 99%

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

Zhang

et al. 2010

Indian J Clin Biochem

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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.

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“…To date, recombinant leptin has been produced essentially in E. coli, necessitating a multistep extraction process that generally includes disruption of the cells, centrifugation, extraction, and refolding of the denatured protein (21,31,57). Although in some cases leptin could be extracted in a soluble form from the E. coli periplasm, the recovery procedure involved osmotic shock and several centrifugation steps (26). In addition, all the methods described to date for recombinant leptin production require purification steps before the leptin's biological activity can be evaluated.…”

Section: Discussionmentioning

confidence: 99%

Effects of Intranasal Administration of a Leptin-Secreting Lactococcus lactis Recombinant on Food Intake, Body Weight, and Immune Response of Mice

Bermúdez‐Humarán

Nouaille

Zilberfarb

et al. 2007

Appl Environ Microbiol

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Leptin is an adipocyte-derived pleiotropic hormone that modulates a large number of physiological functions, including control of body weight and regulation of the immune system. In this work, we show that a recombinant strain of the food-grade lactic acid bacterium Lactococcus lactis (LL-lep) can produce and efficiently secrete human leptin. The secreted leptin is a fully biologically active hormone, as demonstrated by its capacity to stimulate a STAT3 reporter gene in HEK293 cells transfected with the Ob-Rb leptin receptor. The immunomodulatory activity of leptin-secreting L. lactis was evaluated in vivo by coexpression with the human papillomavirus type 16 E7 protein. In C57BL/6 mice immunized intranasally with a recombinant L. lactis strain coproducing leptin and E7 antigen, the adaptive immune response was significantly higher than in mice immunized with recombinant L. lactis producing only E7 antigen, demonstrating adjuvanticity of leptin. We then analyzed the effects of intranasally administered LL-lep in obese ob/ob mice. We observed that daily administration of LL-lep to these mice significantly reduced body weight gain and food intake. These results demonstrate that leptin can be produced and secreted in an active form by L. lactis and that leptin-producing L. lactis regulates in vivo antigen-specific immune responses, as well as body weight and food consumption.

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Efficient Secretion of Biologically Active Recombinant OB Protein (Leptin) inEscherichia coli,Purification from the Periplasm and Characterization

Cited by 33 publications

References 36 publications

Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

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

Effects of Intranasal Administration of a Leptin-Secreting Lactococcus lactis Recombinant on Food Intake, Body Weight, and Immune Response of Mice

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