Publications (7)10 Total impact

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    No preview · Dataset · Aug 2014
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    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Full-text · Article · Apr 2014 · Biotechnology Letters
  • Limei Ren · Xiaofang Cao · Peng Geng · Fang Bai · Gang Bai
    [Show abstract] [Hide abstract] ABSTRACT: In humans, both the N-terminal catalytic domain (NtMGAM) and the C-terminal catalytic domain (CtMGAM) of small intestinal maltase glucoamylase (MGAM) are α-glycosidases that catalyze the hydrolysis of α-(1→4) glycosidic linkages in the process of starch digestion, and are considered to be the main therapeutic targets for type 2 diabetes. In this work, recombinant human CtMGAM has been cloned for the first time, and this, combined with the expression of NtMGAM in Pichia pastoris, made it possible for us to study the catalytic mechanism of MGAM in a well-defined system. The enzymatic kinetic assays of the two catalytic domains suggest that CtMGAM has the higher affinity for longer maltose oligosaccharides. Kinetic studies of commercially-available drugs such as 1-deoxynojirimycin (DNJ), miglitol, voglibose, and acarbose along with a series of acarviosine-containing oligosaccharides we isolated from Streptomyces coelicoflavus against NtMGAM, CtMGAM, and human pancreatic α-amylase (HPA) provide us an overall profile of the inhibitory ability of these inhibitors. Of all the inhibitors used in this paper, DNJ was the most effective inhibitor against MGAM; the K(i) values for the two catalytic domains were 1.41 and 2.04 μM for NtMGAM and CtMGAM, respectively. Acarviostatins 2-03 and 3-03 were the best inhibitors against HPA with relatively high inhibitory activity against CtMGAM. The acarviostatins 2-03 and 3-03 inhibition constants, K(i), for HPA were 15 and 14.3 nM, and those for CtMGAM were 6.02 and 6.08 μM, respectively. These results suggest that NtMGAM and CtMGAM differ in their substrate specificities and inhibitor tolerance despite their structural relationship.
    No preview · Article · Dec 2011 · Carbohydrate research
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    [Show abstract] [Hide abstract] ABSTRACT: Human maltase-glucoamylase (MGAM) hydrolyzes linear alpha-1,4-linked oligosaccharide substrates, playing a crucial role in the production of glucose in the human lumen and acting as an efficient drug target for type 2 diabetes and obesity. The amino- and carboxyl-terminal portions of MGAM (MGAM-N and MGAM-C) carry out the same catalytic reaction but have different substrate specificities. In this study, we report crystal structures of MGAM-C alone at a resolution of 3.1 Å, and in complex with its inhibitor acarbose at a resolution of 2.9 Å. Structural studies, combined with biochemical analysis, revealed that a segment of 21 amino acids in the active site of MGAM-C forms additional sugar subsites (+ 2 and + 3 subsites), accounting for the preference for longer substrates of MAGM-C compared with that of MGAM-N. Moreover, we discovered that a single mutation of Trp1251 to tyrosine in MGAM-C imparts a novel catalytic ability to digest branched alpha-1,6-linked oligosaccharides. These results provide important information for understanding the substrate specificity of alpha-glucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.
    Full-text · Article · Oct 2011 · Protein & Cell
  • Xizhen Qi · Limei Ren · Fang Zheng · Qi Zhang · Fang Bai · Gang Bai
    [Show abstract] [Hide abstract] ABSTRACT: Targeting the important enzyme in human glucose metabolic pathway, we established a high throughput screening model for human pancreatic alpha-amylase inhibitors. Pichia pastoris expression system was used to clone and express the human pancreatic alpha-amylase; we established the alpha-amylase inhibitor screening model using the catalytic properties of enzyme; this model was applied in screening of actinomycete' metabolites; the taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree. We cloned and expressed the intact gene of human pancreatic alpha-amylase successfully; the high-throughput screening model of alpha-amylase inhibitors was established; nearly 2000 actinomycete' metabolites were screened, 14 alpha-amylase inhibitor producing strains were obtained finally, and showed taxonomically rich diversity. The alpha-amylase inhibitor high-throughput screening model had high practical value for developing new hypoglycemic drugs.
    No preview · Article · Aug 2011 · ACTA MICROBIOLOGICA SINICA
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    [Show abstract] [Hide abstract] ABSTRACT: Human pancreatic α-amylase (HPA) catalyzes the hydrolysis of α-d-(1,4) glycosidic linkages in starch and is one of the major therapeutic targets for type II diabetes. Several acarviostatins isolated from Streptomyces coelicoflavus var. nankaiensis previously showed more potent inhibition of HPA than acarbose, which has been successfully used in clinical therapy. However, the molecular mechanisms by which acarviostatins inhibit HPA remains elusive. Here we determined crystal structures of HPA in complexes with a series of acarviostatin inhibitors (I03, II03, III03, and IV03). Structural analyses showed that acarviostatin I03 undergoes a series of hydrolysis and condensation reactions in the HPA active site, similar to acarbose, while acarviostatins II03, III03, and IV03 likely undergo only hydrolysis reactions. On the basis of structural analysis combined with kinetic assays, we demonstrate that the final modified product with seven sugar rings is best suited for occupying the full active site and shows the most efficient inhibition of HPA. Our high resolution structures reported here identify first time an interaction between an inhibitor and subsite-4 of the HPA active site, which we show makes a significant contribution to the inhibitory effect. Our results provide important information for the design of new drugs for the treatment of type II diabetes or obesity.
    Full-text · Article · Nov 2010 · Journal of Structural Biology
  • Peng Meng · Xizhen Qi · Fang Zheng · Limei Ren · Fang Bai · Gang Bai
    [Show abstract] [Hide abstract] ABSTRACT: Targeted at the important enzyme in human glucose metabolic pathway, the purpose of this paper is to establish alpha-glucosidase inhibitors high throughput screening model. Pichia pastoris expression system was used to clone and express the human alpha-maltase glucosidase. Using the catalytic properties of enzyme to establish alpha-glucosidase inhibitor screening model. This model was applied in screening of actinomycete metabolites library. The taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree. The N-terminal catalytic domain of human alpha-maltase glucosidase was successfully cloned and expressed for the first time. The high-throughput screening model of alpha-glucosidase inhibitors was established. A natural product library containing metabolites from nearly 2000 actinomycetes was screened, 20 alpha-maltase glucosidase inhibitor producing strains were obtained finally, of which, 19 strains initially identified as Streptomyces, and showed taxonomically rich diversity. The alpha-glucosidase inhibitor high-throughput screening model has high practical value, this work laid the foundation for developing new hypoglycemic drugs.
    No preview · Article · Aug 2010 · ACTA MICROBIOLOGICA SINICA