Expression of Human Globular Adiponectin-Glucagon-Like Peptide-1 Analog Fusion Protein and Its Assay of Glucose-Lowering Effect In Vivo

Zhao, Tongfeng; Lv, Jing; Zhao, Jiangpei; Huang, Xiao; Xiao, Haijuan

doi:10.7150/ijms.8.203

Cited by 4 publications

(1 citation statement)

References 15 publications

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“…There is considerable evidence suggesting that N-terminal fusion His tags cause a drop in peptides' activities (Huang et al, 2008;Zhao et al, 2011). By contrast, other studies suggest that the N-terminal is not crucial for the activities of fusion proteins (Franklin and Clarke, 2001;Wu et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Expression and purification of porcine PID1 gene in Escherichia coli

Wang¹,

Chen²,

Huang³

et al. 2014

Turk J Biol

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In this study, in order to scale up the production of recombinant porcine phosphotyrosine interaction domain containing 1 (pPID1), a pET-28a (+)-pPID1 expression plasmid was constructed and transformed into Escherichia coli Rosetta (DE3). The recombinant pPID1 was then purified and identified by western blotting, and was also analyzed in vitro for its function. The recombinant protein was tagged with only a His6 tag at its C-terminus, which could be conveniently purified by affinity column. The protein could be induced for efficient expression with 0.75 mM IPTG for 8 h at 30 °C, yielding approximately 3 mg/L. In vitro biological activity assay demonstrated that the refolded purified recombinant pPID1 increased 3T3-L1 preadipocyte proliferation. This study provides a reliable technique for the recombinant expression and purification of pPID1 proteins.

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Section: Discussionmentioning

confidence: 99%

Expression and purification of porcine PID1 gene in Escherichia coli

Wang¹,

Chen²,

Huang³

et al. 2014

Turk J Biol

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A strategy for fusion expression and preparation of functional glucagon-like peptide-1 (GLP-1) analogue by introducing an enterokinase cleavage site

Liu

Ren

et al. 2014

Biotechnol Lett

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KGLP-1, a 31-amino acid glucagon-like peptide-1 (GLP-1) analogue, has a great therapeutic potential for anti-diabetes. In this work, a strategy for expression and purification of functional KGLP-1 peptide has been established. KGLP-1 cDNA was fused with glutathione S-transferase (GST), with an enterokinase cleavage site in the fusion junction. The recombinant fusion protein GST-KGLP-1 was affinity purified via the GST-tag, and then digested with enterokinase. The resulting GST part as well as the enzymes were eliminated by ultra-filtration followed by size exclusion chromatograph. The yield of purified KGLP-1 was approximately 12.1 mg/L, with purity of 96.18 %. The recombinant KGLP-1 was shown to have similar bioactivity as native GLP-1 when evaluated in a Chinese hamster ovary cell line expressing a GLP-1 receptor-egfp reporter gene.

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Alleviation of high-fat diet-induced atherosclerosis and glucose intolerance by a novel GLP-1 fusion protein in ApoE−/− mice

et al. 2016

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We have previously constructed an engineered anti-diabetic fusion protein using glucagon-like peptide-1 and the globular domain of adiponectin. Herein, we evaluated the therapeutic effects of this fusion protein (GAD) on high-fat diet (HFD)-fed ApoE(-/-) mice. The lipid-lowering effect of GAD was determined in C57BL/6 mice using a lipid tolerance test. The effects of GAD on HFD-induced glucose intolerance, atherosclerosis, and hepatic steatosis were evaluated in HFD-fed ApoE(-/-) mice using glucose tolerance test, histological examinations and real-time quantitative PCR. The anti-inflammation activity of GAD was assessed in vitro on macrophages. GAD improved lipid metabolism in C57BL/6 mice. GAD treatment alleviated glucose intolerance, reduced blood lipid level, and attenuated atherosclerotic lesion in HFD-fed ApoE(-/-) mice, which was associated with a repressed macrophage infiltration in the vessel wall. GAD treatment also blocked hepatic macrophage infiltration and prevented hepatic inflammation. GAD suppressed lipopolysaccharide-triggered inflammation responses on macrophages, which can be abolished by H89, an inhibitor of protein kinase A. These findings demonstrate that GAD is able to generate a variety of metabolic benefits in HFD-fed ApoE(-/-) mice and indicate that this engineered fusion protein is a promising lead structure for anti-atherosclerosis drug discovery.

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Expression of Human Globular Adiponectin-Glucagon-Like Peptide-1 Analog Fusion Protein and Its Assay of Glucose-Lowering Effect In Vivo

Cited by 4 publications

References 15 publications

Expression and purification of porcine PID1 gene in Escherichia coli

Expression and purification of porcine PID1 gene in Escherichia coli

A strategy for fusion expression and preparation of functional glucagon-like peptide-1 (GLP-1) analogue by introducing an enterokinase cleavage site

Alleviation of high-fat diet-induced atherosclerosis and glucose intolerance by a novel GLP-1 fusion protein in ApoE−/− mice

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