Mingming Gao

A site-specific PEGylated analog of exendin-4 with improved pharmacokinetics and pharmacodynamics in vivo

Objectives  Our aim was to improve the in vivo pharmacokinetics and pharmacodynamics of exendin‐4 by using site‐specific PEGylation.

Intein-mediated expression and purification of an analog of glucagon-like peptide-1 in Escherichia coli.

To facilitate expression and purification of an analog of GLP-1 (mGLP-1), an intein system was employed in this study. A recombinant fusion protein, CBD-DnaB-mGLP-1, was constructed and expressed in the form of inclusion body. After refolding, the intein-mediated self-cleavage was triggered by pH and temperature shift. By using chitin beads column followed by single step purification, about 2.58 mg of mGLP-1 with the purity of up to 98% could be obtained from 1 L medium. Tricine-SDS-PAGE, RP-HPLC, and ESI-MS were undertaken to determine the purity and molecular weight of mGLP-1. The glucose-lowering activity of mGLP-1 was also preliminarily determined.

Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein

Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.

Improving the anti-diabetic activity of GLP-1 by fusion with globular adiponectin.

Abstract Glucagon-like peptide-1 (GLP-1) as an endogenous glucose-lowering peptide has great potential in diabetes therapy, but its clinical utility is compromised by its limited activity in vivo. Herein to improve the anti-diabetic activity of GLP-1, we constructed a fusion protein (GLP-1-globular adiponectin, GAD) using this peptide and the globular adiponectin. Using recombinant expression, we prepared the GAD fusion protein with a purity of above 95%. In normal mice, we validated the acute glucose-lowering activity of GAD by performing intraperitoneal glucose tolerance test (IPGTT). After that, we evaluated the anti-diabetic activity of this fusion protein in a multiple-low-dose streptozotocin (STZ)-induced diabetic mice model. In this diabetic mice model, GAD treatment greatly reduced the elevated fasting glucose and improved their impaired glucose homeostasis as judged by oral glucose tolerance test (OGTT). After treatment, the fasting glucose was 15.34 ± 2.07 mmol/L and 9.47 ± 1.08 mmol/L for GLP-1-treated and GAD-treated diabetic mice, respectively. Moreover, GLP-1 treatment and GAD treatment improved the pancreas function of the diabetic mice by ~1.5-fold and ~4.2-fold, respectively. Furthermore, GAD treatment greatly reduced the tissue damage in the pancreas of the diabetic mice. These data suggest that GAD possesses an improved anti-diabetic activity in vivo compared with native GLP-1, and therefore it could be regarded as a potential candidate for the future development of anti-diabetic drugs.

Optimized Soluble Expression and Purification of an Aggregation-prone Protein by Fusion Tag Systems and On-column Cleavage in Escherichia coli

Previously we constructed a fusion protein based on GLP-1 and globular adiponectin but unfortunately its yield was low because it was mainly expressed as inclusion bodies. Herein to optimize the soluble expression of this fusion protein we tried several fusion tag systems. Fusion tags, including GST-, Trx- and MBP-tag, greatly improved the soluble expression of the fusion protein. However, these tag-fusion proteins were aggregation-prone as judged by Native PAGE and gel filtration chromatography, and this aggregation reduced the specificity of enterokinase-mediated enzyme cleavage which was essential to remove the fusion tags. To improve the specificity of protein cleavage, we employed on-column cleavage for downstream purification. Finally using optimized expression followed by on-column cleavage, we obtained the product fusion protein with a yield of 1.2 mg per g wet bacterial cells which was 8-fold higher than before. This method improved the yield and simplified the process, and as a convenient method it can also be used for the preparation of other aggregation-prone proteins.

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

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.

PEGylation-aided refolding of globular adiponectin

Globular adiponectin (GAD) as the active domain of adiponectin is a promising candidate for anti-diabetic drug development. The recombinant production of GAD in Escherichia coli, however, is difficult because it is mainly expressed as inclusion bodies which need to be refolded to regain function. In this study we developed a novel method for refolding of GAD with a high efficiency by using polyethylene glycol (PEG) conjugation. An artificially designed DNA sequence encoding for GAD was synthesized and inserted into the pET28a vector to construct an expression plasmid which was thereafter transformed into E. coli BL21 (DE3) host cells for heterologous expression. After bacterial cell culture employing auto-induction medium, the inclusion bodies were collected, washed and dissolved in guanidine hydrochloride before PEG conjugation. Then the PEG-conjugated GAD was refolded by dialysis and purified by two steps of chromatography. The refolded conjugate showed a marked glucose-lowering activity in mice, demonstrating that it had been successfully refolded. As a convenient method, PEGylation-aided refolding could also be tested on other proteins to explore its suitability.

A mutated glucagon‐like peptide‐1 with improved glucose‐lowering activity in diabetic mice

The aim of this study was to characterize the conformation and potency of a mutated glucagon‐like peptide‐1 (mGLP‐1), and evaluate its glucose‐lowering activity in diabetic mice.

Development of a C-Terminal Site-Specific PEGylated Analog of GLP-1 with Improved Anti-Diabetic Effects in Diabetic Mice

Preclinical Research

Vascular endothelial growth factor-B: Impact on physiology and pathology

ABSTRACT Angiogenesis plays an important role in controlling tissue development and maintaining normal tissue function. Dysregulated angiogenesis is implicated in the pathogenesis of a variety of diseases, particularly diabetes, cancers, and neurodegenerative disorders. As the major regulator of angiogenesis, the vascular endothelial growth factor (VEGF) family is composed of a group of crucial members including VEGF-B. While the physiological roles of VEGF-B remain debatable, increasing evidence suggests that this protein is able to protect certain type of cells from apoptosis under pathological conditions. More importantly, recent studies reveal that VEGF-B is involved in lipid transport and energy metabolism, implicating this protein in obesity, diabetes and related metabolic complications. This article summarizes the current knowledge and understanding of VEGF-B in physiology and pathology, and shed light on the therapeutic potential of this crucial protein.