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Preparation, Modification and Bioactivity of a Derivative of Exendin-4

Author: WangXiaoXi
Tutor: WuZiRong
School: East China Normal University
Course: Biochemistry and Molecular Biology
Keywords: Excndin - 4 derivatives Cloning and expression Purification Modify Biological Activity
CLC: R914
Type: Master's thesis
Year: 2011
Downloads: 51
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Abstract

Exendin-4 is by a polypeptide consisting of 39 amino acids in Americas Gila monster (Heloderma suspectum), salivary gland secretion in mammals by -1 (glucagon-like peptide-1, GLP-1), glucagon-like peptide- receptor binding role, is a GLP-1 receptor agonist. Exendin-4 and GLP-1 receptor binding, may promote insulin secretion when the glucose concentration is too high in the body, thus effectively reducing the blood glucose concentration; not have this effect when the blood glucose is low, it will not cause hypoglycemia. , Exendin-4 may also be enhanced insulin sensitivity, and inhibition of beta-cell apoptosis, and promote the p-cell regeneration, stimulate the proliferation of p-cells, and Exendin-4 itself has a strong stability, resistant to body dipeptide yl peptidase IV degradation. These features make Exendin-4 treatment in type 2 diabetes drug development has broad prospects. In this study on the basis of the sequence of Exendin-4, the design of a new type of Exendin-4 derivatives (E306) sequence, E306 polypeptide prepared by genetic engineering methods. Animals detected in vivo activity after recycling of Biotin-NHS modified E306 lysine, to make it suitable for oral administration. Contents and results include the following three parts: 1 E306 polypeptide cloning, expression and purification of Exendin-4 structure-activity relationships, Exendin-4 before the 30 amino acid sequence of Exendin-4 (1-30) as a starting point, the added at the end of the six lysine, E306 sequence design. The design of PCR primers, 6-lysine was added in the downstream primer corresponding to nucleotides. E306 gene, obtained by PCR with the plasmid pET-32a containing the gene sequence of Exendin-4 (1-30) ()-E30 as a template. E306 gene was cloned into the vector pET-32a (), will be constructed in the recombinant expression vector pET-32a ()-E306, transformed into E. coli BL21 (DE3) for expression of genetic engineering strains pET-32a () -E306/BL21 (DE3) . By inducing expression in the relative molecular weight of 21 kDa appears purpose of fusion protein band, and the expression in the form of soluble expression, the expression level of 22.72%. The fusion protein by affinity chromatography, by enterokinase digestion and secondary affinity chromatography and, finally obtained the electrophoretically pure E306 polypeptide calculated E306 polypeptide production of 0.64mg / g microbial cells. E306 polypeptide Properties of Kunming mice by intraperitoneal injection E306 polypeptide, glucose tolerance test results show that peptides E306 can significantly improve the mouse glucose tolerance, reduce blood sugar caused due to the injection of glucose concentration, 2 h the average glucose level less than the control group, 41% (P lt; 0.001). Detected in the diabetes model mice (db / db mouse) E306 polypeptide activity, and found that the the E306 can significantly improve the glucose tolerance of db / db mouse on within 2 hours, with an average blood glucose level is lower than the control group, 48% (P lt ; 0.001). Kunming mice in vivo assays intraperitoneal injection of E306 polypeptide hypoglycemic duration, results measured 1 nmol / kg dose, hypoglycemic polypeptide E306 duration of up to 6 h. E306 polypeptide the Kunming mice hypoglycemic duration with increasing dose prolonged when the dose increased by 0.1 nmol / kg to 10 nmol / kg when, corresponding to the duration of hypoglycemic 1h extended to 7 h. Circular dichroism determination the E306 secondary structure of the polypeptide, E306 polypeptide secondary structure composition and content: alpha-helix (68.8%), p-angle (1.9%) (29.3% random coil) ; E30 peptide secondary structure were measured under the same conditions, the composition and content of the secondary structure: alpha-helix (39.8%), beta-corner (7.4%) (52.8%), random coil. E306 polypeptide than the E30 polypeptide alpha-helix content increased by 29%, the added six lysine to increase the E306 polypeptide alpha-helix content. E306 polypeptide Biotin-NHS-modified and modified products activity of the E306 peptide Biotin-NHS molar ratio of 1:8 mixed at room temperature for 1 h, Tricine-SDS-polyacrylamide gel electrophoresis and HPLC The analysis results show that the Biotin-NHS bonded to E306 polypeptide molecules. Modified products alone E306 polypeptide healthy mice a dose of 300 nmol / kg, respectively, given by way of oral glucose tolerance test results show that: the to give modified product group had significantly improved glucose tolerance in mice, two groups mice within 1 h average blood glucose values ??were lower than the control group, 38% (P lt; 0.01), 21% (P gt; 0.05) In summary, this study constructed expression vector pET-32A ()-E306, and transformed into E. coli BL21 (DE3) pET-32a () -E306/BL21 (DE3), a genetically engineered bacteria. E306 polypeptide purified after intraperitoneal injection, can also significantly improve the glucose tolerance of healthy mice given a glucose load, and diabetic model mice (db / db mouse); mice drop in the low dosage of 1 nmol / kg, sugar duration of up to 6 h. Biotin-NHS modified lysine E306 polypeptide than before modification of the modified product showed good oral hypoglycemic effect. The above results for the treatment of diabetes drug research and development to provide a reference.

Preparation, Modification and Bioactivity of a Derivative of Exendin-4

Abstract

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