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Method for preparing parathormone 1-34

A technology of parathyroid hormone and fusion protein, which is applied in the direction of preparation methods of parathyroid hormone and peptides, botany equipment and methods, etc., and can solve problems such as complex preparation processes

Inactive Publication Date: 2007-02-21
DONGGUAN TAILI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the need to use double enzyme digestion, the preparation process disclosed in this patent application is also relatively complicated
[0005] It has long been known that thioredoxin (Thiroedoxin, sometimes referred to as "Trx") is a protein commonly found in yeast, bacteria, animals, and plants. This protein is also a commonly used PTH (1-34) expression host, such as Endogenous protein of yeast or E. coli, but so far no one has used this protein to fuse with PTH(1-34) to easily produce hPTH(1-34)

Method used

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  • Method for preparing parathormone 1-34
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  • Method for preparing parathormone 1-34

Examples

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Embodiment 1

[0035] Example 1 Design and artificial synthesis of DNA sequences expressing hPTH(1-34)

[0036] According to the amino acid sequence of hPTH(1-34) (see Table 1) and the codon preference of Escherichia coli, an optimized DNA sequence suitable for expression in Escherichia coli was artificially synthesized. When synthesizing the hPTH (1-34) gene, the Kpn I restriction site GGTACC was introduced at the 5' end of the gene, the stop codon TAA and the Sal I restriction site GTCGAC were introduced at the 3' end, and the The coding sequence GACGACGACGACAAG of the enterokinase digestion recognition site is added behind the Kpn I restriction site to obtain sequence 1 (see Figure 10 ). To facilitate subsequent subcloning, the synthetic gene was cloned into pUC18 for preservation of the synthetic DNA sequence. Plasmid pUC18 contains the same Kpn I and Sal I restriction sites as plasmid pET32a(+).

[0037] serial number

[0038] 15

[0039] NOTE: The amino acid s...

Embodiment 2

[0040] The construction process of embodiment 2 recombinant plasmids

[0041] The following molecular cloning operation methods, unless otherwise specified, refer to the literature: Molecular Cloning Experiment Guide (translated by Huang Peitang et al., [US] Sambrook et al., Science Press, 2002). The DNA extraction kit (UNIQ-10), DNA gel recovery kit (UNIQ-10) and ligation kit used in DNA manipulation were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. The cloning vector pUC18 and restriction endonucleases were purchased from Fermentas LifeScience Company. The expression vector pET32a(+), Escherichia coli TOP10 and BL21(DE3) were purchased from Novagen. The Escherichia coli host used for cloning is TOP10, and the Escherichia coli host used for expression is BL21(DE3). The genotype of BL21 (DE3) is: hsdS gal (λcIts857ind1 Sam7 nin5 lacUV5-T7 geneI). BL21(DE3) has a T7 RNA polymerase gene, and under the induction of IPTG, a large amount of T7 RNA p...

Embodiment 3

[0064] Induced expression of embodiment 3 engineering bacteria

[0065] Escherichia coli BL21(DE3) is transformed with the recombinant plasmid pET-PTH(1-34) DNA, and the obtained is the genetically engineered bacteria for expressing the rhPTH(1-34) fusion protein. Pick 8 single colonies, culture overnight at 37°C in 5ml LB medium (1% peptone, 0.5% yeast extract, 1% NaCl) containing 100μg / ml Amp, transfer to 50ml containing 100μg / ml Amp cultured in LB medium at 37°C, and the remaining bacterial solution was subpackaged with 15% glycerol and frozen. OD 600 When it reached 0.5, IPTG was added to a final concentration of 0.5mM to induce expression, and samples were taken for SDS-PAGE electrophoresis after 4 hours. Compared with the uninduced control, the induced recombinants all had an expression band with an expected molecular weight of about 20 kDa, and the expression amount was more than 25% of the total bacterial protein. Bacteria No. 6 with the highest yield was taken as ...

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Abstract

This invention provides a method for preparing recombinant human parathyroid hormone 1-34 (rhPTH (1-34)). The method comprised: (1) expressing with an expression vector capable of expressing a fusion protein with an amino acid sequence (from N-terminal to C-terminal) containing thioredoxin, (His) 6, enterokinase recognition site and parathyroid hormone 1-34 peptide; (2) purifying the fusion protein by nickel ion complexation affinity chromatography; (3) cutting the purified fusion protein with enterokinase to release the rhPTH (1-34) from the fusion protein. By this method, rhPTH (1-34) can be purified rapidly, simply and efficiently, and the recovery yield is largely increased.

Description

technical field [0001] The invention relates to protein preparation technology, in particular to a method for preparing human parathyroid hormone 1-34 (hPTH(1-34)) by using genetic engineering technology. Background technique [0002] Parathyroid hormone (Parathyroid Hormone, PTH) is synthesized by the parathyroid hormone chief cell and consists of 84 amino acids. It is increasingly recognized that PTH can increase the number of osteoblasts, which are very active in synthesizing new bone matrix, and can alter gene expression in bone in vivo. PTH also has the functions of lowering blood pressure, regulating the expression of vitamin D receptor (VDR), and regulating the activity of alkaline phosphatase. Tregear et al. (Tregear GW et al., Endocrinology, 1973, 93:1349) proved by experiments that only 1-34 amino acid residues at the amino terminal are required for PTH to play the role of calcium and phosphorus regulating molecules. However, PTH(1-34) does not contain cysteine ​...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/62C12N15/12C07K19/00C07K14/635C07K1/14C12N15/70C12N1/21
CPCC07K2319/50C07K2319/00C07K2319/21C12N9/0036C07K14/635
Inventor 杨立明张仁怀刘社际阳勇何凯黄碧莲刘德林
Owner DONGGUAN TAILI BIOTECH
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