The Study on the Construction, Expression and Activity of Exendin-4/HSA Long-acting Fusion Protein
|Keywords||Bglbrick Exendin-4/HSA fusion protein synthetic biology|
Diabetes mellitus is a group of metabolic diseases, mainly including type1(insulin-dependent) diabetes and type2(non-insulin-dependent) diabetes. It is predicted that the number of diabetics all over the world will reach300million by2025,90%of which are type2diabetes. Traditional treatments for type2diabetes mainly include metformin, sulfonylureas. Anyhow, long-term use of these drugs can lead to islet β cell dysfunction and the progression of the disease cannot be effectively controlled.Glucagon-like peptide-1(GLP-1) is a peptide hormone secreted by the intestinal L cells. It can stimulate insulin secretion, improve the biosynthesis of the insulin and affect glucose metabolism by regulating the secretion of the insulin and glucagon. GLP-1has very good performance in curing diabetes mellitus.Exendin-4(E4) is a hormone substance consisting of39amino acids, which was found in the saliva of Gila monster living in the Southwest America and Mexico desert. It has53%similarity with GLP-1in structure and almost the same biological effects as GLP-1does. It can increase intracellular cAMP concentration, stimulate the secretion of glucose-dependent insulin, inhibit β-cell apoptosis and stimulate β-cell proliferation and neogenesis. It can also weaken biological effects like hunger, postprandial glucose concentrations and glucagon concentrations. The half-life of E4is2to3hours, which is much longer than that of GLP-1(1-2min), E4also shows significantly higher stability, biological activity and better therapeutic effect than GLP-1. As therapeutic agents for diabetes its half-life is still short and the content of natural product is relatively low. Besides the chemical synthesis method has many disadvantages like high cost, multiple steps, low yield and hazardous chemicals involution the synthesis process. All of these cannot meet the massive clinical needs. Related studies show that the fusion protein constructed by genetic engineering methods can help to prolong the half-life of Exendin-4and greatly reduce the production cost.The purpose of this experiment is to achieve fusion expression of optimized Exendin-4gene and HSA gene in different ways by BglBrick method in synthetic biology and screen candidates for the long-acting fusion protein by bioactivity testing in vitro. It’s expected that the long-acting fusion protein scan prolong their half-life and improve the therapeutic effects on condition that they will show high biological activity. The main contents and results are included as follows.1. pHI_D2_HSA/IFN plasmid was used as the template. HSA gene fragments of the corresponding restriction sites were obtained by PCR amplification, in which Xho I, BglⅡ, Not Ⅰ and BamH Ⅰ restriction sites were added in the upstream and downstream primers, and inserted into the expression vector plasmid pPICZaA, for the construction of pPICZaA-HSA expression vector.2. The open reading frame sequence for encoding Exendin-4was retrieved in NCBI database. After optimization according to the preference of Pichia pastoris codon, at both terminals of fragment were added Xho I, Bgl Ⅱ and BamH Ⅰ and Not I restriction sites separately. The fragment was introduced in the expression vector plasmid pPICZaA by artificial synthesis for the construction of pPICZaA-E4expression vector.3. Ten kinds of HSA/Exendin-4fusion protein expression vectors were obtained by using Bglbrick Method synthetic biology. pPICZaA-HSA and pPICZaA-E4expression vector, as the basis of two expression vectors, constructed different forms of Exendin-4/HSA fusion protein expression vectors. The expression vectors were linearized before integrated into the chromosome of Pichia pastoris KM71H by electro transformation. Ten corresponding target proteins were then successfully expressed after induction of methanol and SDS-PAGE analysis of the obtained supernatant broth.4. The purity of achieved target proteins of eight was above90%by the use of hydrophobic chromatography column, ion exchange chromatography or affinity chromatography. These techniques were applied in the separation and purification of the supernatant in the expressed broth after induction step by step.5. GLP-1protein was used as the positive drug to test its effect on the proliferation of RIN-m-5F and MIN6cells by MTT method, on the amount of insulin secreted by INS-1and MIN6cells and on the level of cAMP secreted by BHK-GLP-1R cells by ELISA method. After a comprehensive comparison of the above methods, the method for the detection of biological activity in vitro was finally established, in which ELISA was applied to test the effect of target proteins on the amount of insulin secreted by INS-1cells.This method is simple, reproducible, and suitable for in vitro biological activity detection of the fusion protein.6. The aboved screened method was used in the evaluation of in vitro biological activity of the fusion protein. The final concentration of eight fusion proteins was250μg/mL and treated on the MIN6cells in a logarithmic phase. Two hours later, insulin ELISA kit was used to detect the amount change in insulin secretion. The experimental results show that long-acting fusion protein of eight on MIN6cells have a stimulating insulin secretion effect, which (E4)4-HSA, E4-HSA-E4fusion protein have the best insulin secretion effect, increased by41.06%,42.89%.In the experiments long-term fusion protein expression vectors reconstructed by BglBrick way in synthetic biology and the rapid assembly of Exendin-4at the different terminals of HSA has been achieved. The expression vectors were integrated into the chromosome of Pichia pastoris KM71H by electro transformation and then successfully expressed ten corresponding fusion proteins after induction of methanol. It provides a new idea for the rapid construction of fusion proteins. Fusion proteins with high purity were achieved by the separation and purification of supernatant in the expressed broth after induction which facilitates in vivo bioactivity tests of fusion protein. The screened detection method achieved good results, established good reproducibility and was applicable to in vitro bioactivity detection of the fusion protein. The evaluation result of bioactivity of the fusion protein in vivo builds a foundation for subsequent bioassays of biological activity in vitro.