Exendin-4 analogue fusion protein and preparation method and application thereof
A technology of fusion proteins and analogs, which can be used in drug combinations, peptide/protein components, chemical instruments and methods, etc., can solve the problems of no clinical use value, cytotoxicity and complement activation damage, and small molecular weight. Prolonged circulating half-life in vivo, prolonged functional half-life in vivo, well-tolerated effect
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Embodiment 1
[0052] Embodiment 1, construct the expression plasmid encoding Ex(1-45)-L-vFc fusion protein
[0053] The gene sequence encoding α1 microglobulin (α1microglobulin) leader peptide and mature Ex(1-45) polypeptide, the 10 amino acid flexible peptide linker (GlyGlyGlyGlySerGlyGlyGlyGlySer) and the fusion gene of human IgG2Fc variant (Pro331Ser / Thr250Gln / Met428Leu mutation) are all It is an artificially optimized CHO cell preferred codon, the full-length sequence is obtained by chemical synthesis (see the fusion protein nucleotide and amino acid sequence figure 1). In order to facilitate the insertion of the target fragment into the specific site of the expression vector, there is a restriction enzyme endonuclease site at the 5' and 3' ends of the synthesized fragment, respectively SpeI and EcoRI. After verification, the GLP-1 gene was digested with SpeI and EcoRI, and then inserted into the corresponding restriction site of the transformed plasmid pCDNA3.1 to obtain the fusion ge...
Embodiment 2
[0054] Embodiment 2, the expression of fusion protein in transfection cell line
[0055] The recombinant expression vector plasmid was transfected into a mammalian host cell line to express the Ex(1-45)-L-vFc fusion protein. In order to stably express at a high level, the preferred host cell line is DHFR enzyme-deficient CHO-cells (see US Pat. No. 4,818,679). In this example, the host cell line is the CHO-derived cell line DXB11. A preferred method of transfection is electroporation, although other methods including calcium phosphate co-sedimentation, lipofection, and protoplast fusion can also be used. In electroporation, with a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) set to a 240V electric field and a 1050 μFd capacitance, 3×10 cells in a cuvette were used. 7 Add 40 μg of plasmid linearized with PvuI to each cell. Two days after transfection, the medium was changed to growth medium containing 0.6 mg / mL G418. Transfectants were screened for resistan...
Embodiment 3
[0057] Embodiment 3, purification and qualitative of fusion protein
[0058] The conditioned medium containing the fusion protein was titrated to pH 7-8 with 1N NaOH and filtered through a 0.45 micron nitrocellulose filter. The filtrate was loaded onto a Protein A column equilibrated in phosphate buffered saline (PBS). After the fusion protein is bound to the Protein A column, discard the effluent fraction. The column was washed with PBS until the OD value at 280nm was below 0.01. The bound fusion protein was then eluted with 0.1 M citrate buffer, pH 3.75. With 0.4 volume of 1MK 2 HPO 4 For neutralization, fractions containing purified protein were pooled and dialyzed against PBS. Then filter through a 0.22 μm nitrocellulose filter and store at 4 °C. Under non-reducing conditions, the molecular weight of the purified Ex(1-45)-L-vFc protein ranged from 40 to 45 kDa as determined by SDS-PAGE. Under reducing conditions, the purified protein migrates to approximately 90 kDa...
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