Recombinant Expression of Biologically Active Rat Leptin in Escherichia coli

Park, Jung Hyun; Lee, Hyun Hee; Na, Shin Young; Ju, Sung Kyu; Lee, Yun Jung; Lee, Myung Kyu; Kim, Kil Lyong

doi:10.1006/prep.2001.1412

Cited by 14 publications

(8 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cells were seeded in a 96-well plate (Corning) and transfected with the human LEPR overexpression vector pcDNA-Lepr, the control pRL-TK (Promega) and the pGL6-Stat3 plasmid which contains the luciferase gene under control of the STAT3-inducible promoter. One day after transfection, the cells were treated with different concentrations of WT and mutant recombinant rat LEP proteins (encoded by cDNA inserted between BamH I and Sal I sites of pET-21a(+)), which were produced following the previous report62. LEP-induced luciferase activity was measured by Dual-Glo Luciferase Assay System (Promega) following the manufacturer’s manual.…”

Section: Methodsmentioning

confidence: 99%

The 14th Ile residue is essential for Leptin function in regulating energy homeostasis in rat

Shou

Zhu

et al. 2016

Sci Rep

View full text Add to dashboard Cite

LEPTIN (LEP) is a circulating hormone released primarily from white adipocytes and is crucial for regulating satiety and energy homeostasis in humans and animals. Using the CRISPR technology, we created a set of Lep mutant rats that carry either null mutations or a deletion of the 14th Ile (LEP∆I14) in the mature LEP protein. We examined the potential off-target sites (OTS) by whole-genome high-throughput sequencing and/or Sanger-sequencing analysis and found no OTS in mutant rats. Mature LEP∆I14 is incessantly produced and released to blood at a much elevated level due to the feedback loop. Structure modeling of binding conformation between mutant LEP∆I14 and LEPTIN receptor (LEPR) suggests that the conformation of LEP∆I14 impairs its binding with LEPR, consistent with its inability to activate STAT3-binding element in the luciferase reporter assay. Phenotypic study demonstrated that Lep∆I14 rats recapitulate phenotypes of Lep-null mutant rats including obesity, hyperinsulinemia, hepatic steatosis, nephropathy, and infertility. Compared to the existing ob/ob mouse models, this Lep∆I14/∆I14 rat strain provides a robust tool for further dissecting the roles of LEP in the diabetes related kidney disease and reproduction problem, beyond its well established function in regulating energy homeostasis.

show abstract

Section: Methodsmentioning

confidence: 99%

The 14th Ile residue is essential for Leptin function in regulating energy homeostasis in rat

Shou

Zhu

et al. 2016

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Leptin replacement or addition inadequately simulates changes in leptin levels that are associated with exercise, obesity, or disease because it often induces leptin levels outside the physiologically relevant range of variation. Recombinant leptin is available for several model species, including mice ( Barrachina et al 1997), rats (Park et al 2001;Crowley et al 2004), dogs (Iwase et al 2000), pigs (Ramsay et al 1998), and cows (Raver et al 2000), and can be administered to mimic high plasma levels associated with obesity. However, the temporal pattern of leptin secretion is difficult to replicate.…”

Section: Introductionmentioning

confidence: 99%

Leptin Levels and Body Composition of Mice Selectively Bred for High Voluntary Locomotor Activity

Girard¹,

Rezende²,

Garland³

2007

Physiological and Biochemical Zoology

View full text Add to dashboard Cite

Selective breeding produced four replicate lines of high-runner (HR) mice that run on wheels for approximately 2.7 times more revolutions per day than four unselected control lines. Previous studies found that HR mice of both sexes have lower body fat (isotope dilution at 15 wk of age) and that males (females not studied) have smaller retroperitoneal fat pads (17 wk). HR mice also exhibit elevated plasma corticosterone and insulin-stimulated glucose uptake by some hindlimb muscles but apparently do not differ in circulating insulin or glucose levels (males at 18 wk). Given their lower body fat and higher activity levels, we hypothesized that HR mice would have lower circulating leptin levels than controls. Female mice were given wheel access for 6 d at 7 wk of age, as part of the routine wheel testing for the selective breeding protocol, and then were killed after one additional week without wheels to reduce possible acute effects of activity on leptin. As hypothesized, serum leptin levels were significantly lower in HR mice. ANCOVA indicated that leptin was strongly positively correlated with both total body fat (measured by ether extraction) and body mass change from weaning, but HR mice still had significantly lower adjusted leptin levels (ANCOVA). Within HR lines but not within control lines, individual variation in leptin levels was negatively correlated with amount or speed of wheel running measured a week before being killed. Growth from weaning to euthanasia and body dry mass were lower in HR mice than in controls, but absolute dry masses of the ventricles, liver, gut, and uterus plus ovaries did not significantly differ, nor did percentage of the total dry mass as fat. HR mice offer a novel model for studying the causes and consequences of physiologically relevant variations in serum leptin.

show abstract

“…So many researchers have tried to express the recombinant leptin in Escherichia coli. But most of recombinant human leptin expressed in insoluble inclusion bodies (IBs) [6][7][8]. Purification of recombinant, biologically active leptin from E. coli inclusion bodies has also been reported previously.…”

Section: Introductionmentioning

confidence: 92%

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

Zhang

et al. 2010

Indian J Clin Biochem

View full text Add to dashboard Cite

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.

show abstract

Recombinant Expression of Biologically Active Rat Leptin in Escherichia coli

Cited by 14 publications

References 45 publications

The 14th Ile residue is essential for Leptin function in regulating energy homeostasis in rat

The 14th Ile residue is essential for Leptin function in regulating energy homeostasis in rat

Leptin Levels and Body Composition of Mice Selectively Bred for High Voluntary Locomotor Activity

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

Contact Info

Product

Resources

About