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Production of reorganized human parathyroid hormone 1-34 peptide

A parathyroid hormone and precursor peptide technology, applied in the field of DNA recombination, can solve the problem that the production process of PTH (1-34) peptide is not very satisfactory

Inactive Publication Date: 2003-06-18
中国科学院上海生物工程研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, so far, the production process of PTH(1-34) peptide is not very satisfactory

Method used

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  • Production of reorganized human parathyroid hormone 1-34 peptide
  • Production of reorganized human parathyroid hormone 1-34 peptide
  • Production of reorganized human parathyroid hormone 1-34 peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] gene synthesis

[0074] According to the amino acid sequence of the Gly-Ser-Pro-hPTH34 peptide, the preferred codons of Escherichia coli were selected, and the following 6 fragments (P1-P6) were designed and synthesized on the ABI 391 nucleic acid synthesizer:

[0075] P1.5'-cc gga tcc ccg tct gtt tct gaa atc-3' (26bp) (SEQ ID NO: 5)

[0076] P2.5'-cag ctg atg cac aac ctg ggt aaa cac ctg aac tct atg gaa cgt gtt gaa tgg-3' (54bp) (SEQ ID NO: 6)

[0077] P3.5'-ctg cgt aaa aaa ctg cag gac gtt cac aac ttc taa tag c-3' (40bp) (SEQ ID NO: 7)

[0078] P4.5'-cag ctg gat ttc ag-3' (14bp) (SEQ ID NO: 8)

[0079] P5.5'-t acg cag cca ttc a-3' (14bp) (SEQ ID NO: 9)

[0080] P6.5'-cg ctc gag cta tta gaa gtt gtg aac-3' (26bp) (SEQ ID NO: 10)

[0081] P1, P2, and P3 make up the upper chain part of the gene. The 3' end of P4 and P1 and the 5' end of P2 each have 7 bases complementary. The 3' end of P5 and P2 and the 5' end of P3 each have 7 bases complementary. P6 is complementar...

Embodiment 2

[0095] Construction of engineered bacteria

[0096] Then, the target gene constructed in Example 1 was double-digested with BamHI / XholI, ligated with the pGEX-4T-3 plasmid treated in the same way, and transfected into the host strain BL21. Positive clones were screened on the resistance plate, and a single colony was selected for culture. When the cell density reaches 0.6OD 600 , add IPTG with a final concentration of 0.4mM to induce expression screening, select a strain from the positive strains expressing the target product, extract its plasmid, and use the gene fragment P 1 ,P 6 As primers, carry out PCR reaction, and PCR amplified fragments of expected length can be seen. DNA sequence analysis was also in line with expectations (see PCR results and gene sequencing results Figure 1A ). The strain is named as Escherichia coli pGEX-PT, and its expression results are shown in Figure 1B , at a molecular weight of 30,000 Daltons, an obvious fusion protein expression band ...

Embodiment 3

[0099] Preliminary experiment of thrombin digestion and exploration of conditions for PEP digestion

[0100] (1) Exploration of PEP digestion conditions

[0101] In order to determine whether the Gly-Ser-Pro-PTH (1-34) peptide in the designed fusion protein can be successfully processed by proline endonuclease, the chemically synthesized precursor 37 peptide was first used to carry out conditional experiments . The PTH(1-34) peptide precursor 37 peptide was dissolved in 20mM appropriate buffers with pH values ​​of 5.8, 6.8, 7.5, 8.0, 8.5, 9.0, 9.5, and 10 (where pH 5.8 was phosphate buffered saline solution, and the rest of the pH values ​​are Tris-HCl buffer), and made into 1mg / ml substrate reaction solution. PEP enzyme was added at a ratio of 1:50 (w / w). After acting on the substrate for 7 hours at 34 degrees Celsius, HPLC detects the content of 34 peptide products respectively. The experimental results show that the optimum pH for PEP to 37 peptide is 8.5 ( Figure 2A )...

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Abstract

A process for preparing recombinant human parathyroxin 1-34 peptide includes such steps as culturing host cell in proper condition, separating Gly-Ser-Pro-PTH 1-34 peptide, severing by Pro endopeptidase to form PTH 1-34 peptide and separating and purifying PTH 1-34 peptide. The relative coding sequence, carrier and host cell are also disclosed. Its advantages are simple process, and high purity and activity.

Description

technical field [0001] The present invention relates to DNA recombination technology. More specifically, the present invention relates to the production process of human parathyroid hormone 1-34 peptide and the corresponding coding sequence, vector and host cell. Background technique [0002] Human parathyroid hormone is a straight-chain polypeptide hormone secreted by the parathyroid gland, consisting of 84 amino acids, and the N-terminal 1-34 peptide has its complete biological function (Potts, J.T., Jr., Kronenberg, H. M., and Rosenblatt, M. Parathyroid hormone: chemistry, biosynthesis and mode of action (1982) Adv. Protein Chem. 35, 323-396.). [0003] At present, two kinds of PTH-related receptors have been cloned, and the type I receptor isolated from bone cells and kidney cells can mediate a variety of PTH-related signal responses (Hisashi Takasu, Thomas J. Gardella, Michael D Luck, et al. Amino-Terminal Modification of Human Parathyroid Hormone (PTH) Selectively Al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C07K14/635C12N15/63C12P21/02
Inventor 陈常庆修朝阳李民
Owner 中国科学院上海生物工程研究中心
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