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Human pancreas hyperglycemiacin relative peptide-2 analogue

A technology for glucagon and related peptides, which is applied in the direction of glucagon, hormone peptides, animal/human proteins, etc., and can solve the problem of high cost

Inactive Publication Date: 2006-11-15
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, foreign countries mainly use chemical synthesis to prepare short peptides, and the cost is still high. Genetic engineering is used to secrete and express Pro-Pro-h[Gly 2 ]GLP-2, no success yet

Method used

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  • Human pancreas hyperglycemiacin relative peptide-2 analogue
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  • Human pancreas hyperglycemiacin relative peptide-2 analogue

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1Pro-Pro-h[Gly 2 ] Design, synthesis and cloning of GLP-2(1-35) polypeptide gene

[0044] According to codon and design requirements of E. coli, the alanine ( 2 The 35 amino acid sequence of human glucagon-related peptide-2 in which the Ala) residue is replaced by Gly and 2 prolines are extended at the N-terminal is converted into a nucleotide sequence, and its structural framework is shown in FIG. 2 . A BamH I restriction site was added at the 5' end, and a stop codon TAA and a HindIII restriction site were added at the 3' end. After computer-assisted analysis, it was determined that the gene was 124 bp in length and divided into 53, 54, and 53 bases. For the three oligonucleotide fragments, at the same time, a downstream primer M4 was designed for the primary screening of positive clones by PCR.

[0045] Synthesize 4 sequences as follows:

[0046] M1 (5'GCGG G GAT CC G CCG CAC GGT GAC GGT TCT TTC TCT GAC GAA ATG AAC ACC ATC 3’, including BamH I restric...

Embodiment 2

[0053] Embodiment 2Pro-Pro-h[Gly 2 ]Expression of GLP-2(1-35) polypeptide gene in Escherichia coli

[0054] Recombinant expression strain PED-PPGLP-2 / BL21 was shaken overnight at 37°C in liquid LB medium, transferred to corn steep liquor fermentation medium with 2% inoculum, cultured at 37°C for 4 hours, and induced expression with a final concentration of 5 mmol / l lactose , Collect the fermented cells after 4h. Reserved samples were analyzed by 15% SDS-PAGE and thin-layer scanning. An obvious protein band appeared at the molecular weight of about 17700Da ( Figure 1B ), with the fusion protein AnsB-C-Pro-Pro-h[Gly 2 ] The GLP-2 theoretical calculation value is consistent. The fusion protein reached a stable maximum expression level 8 hours after induction, and Densitometric analysis showed that the fusion protein accounted for more than 40% of the total bacterial protein and formed inclusion bodies in the cell.

Embodiment 3

[0055] Embodiment 3 Fusion protein separation and purification

[0056] The engineered bacteria after induced expression were collected by centrifugation, and the bacteria were suspended in the wall-breaking solution (pH8.0 phosphate buffered saline, 0.02% lysozyme). After the bacteria were lysed, inclusion bodies were obtained, and the inclusion bodies were washed, dissolved in urea and The pure fusion protein AnsB-C-Pro-Pro-h [Gly 2 ]GLP-2( Figure 2A ).

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Abstract

A human glucagon associated peptide-2 analog Pro-Pro-h[Gly2]GLP-2(1-35) for treating the short bowel syndrome and malabsorption of stomach and intestine is prepared through configuring genetic engineering bacterium for effective expression of Pro- Pro-h[Gly2]GLP-2(1-35) in colibacillus cell, splitting, washing, dissolving urea, depositing in alcohol, separating fusion protein, hydrolyzing it, chromatography by DEAE-52 column, separating by HPLC, and freeze drying.

Description

technical field [0001] The technology of the present invention is a human glucagon-related peptide-2 analog Pro-Pro-h[Gly 2 ] GLP-2(1-35) peptide. Using the new fusion expression preparation technology platform for active polypeptides with a length of 30-70aa constructed by our laboratory, we constructed a high-efficiency fusion expression of human glucagon-related peptide-2 analogue Pro-Pro-h[Gly 2 ] the genetically engineered bacteria of GLP-2 (1-35), the alanine ( 2 Ala) residues were replaced by Gly, and 2 proline residues were added at the N-terminus. In Pro-Pro-h[Gly 2 ] An acid-sensitive site (Asp-Pro) was designed at the N-terminus of the GLP-2(1-35) peptide, and the fusion protein AnsB-C-Pro-Pro-h[Gly 2 ]GLP-2 obtains the target peptide Pro-Pro-h[Gly 2 ]GLP-2(1-35) peptide, Pro-Pro-h[Gly 2 ]GLP-2(1-35) peptide is cleaved by dipeptidase IV and PPCE enzymes in vivo to generate highly active h[Gly 2 ] GLP-2. The recombinant bacteria were lysed → washed → redissol...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K19/00C12N15/16C12N15/62C12N15/63
Inventor 刘景晶李泰明吴洁
Owner CHINA PHARM UNIV
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