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Production and purification method of teriparatide hPTH (1-34)

A technology of teriparatide and purification method, which is applied in the field of genetic engineering, can solve the problems of high price of teriparatide products, cumbersome purification steps, and low yield, and achieve fewer steps, high yield and purity, and low equipment requirements Effect

Pending Publication Date: 2021-10-26
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current generation and purification method of teriparatide hPTH(1-34) needs to use enzymes to remove the label, and more tedious purification steps need to be used. Various column chromatography techniques, such as affinity chromatography, coagulation chromatography, etc. Gel exclusion chromatography, etc., the yield is low and the cost is high, resulting in high product price of teriparatide

Method used

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  • Production and purification method of teriparatide hPTH (1-34)
  • Production and purification method of teriparatide hPTH (1-34)
  • Production and purification method of teriparatide hPTH (1-34)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Construction of Teriparatide Fusion Protein Expression Constructs Containing Different Intein MtuΔI-CM Mutants The expression vector used in the examples of this application is a fusion protein expression vector containing 4 different MtuΔI-CM mutants (pET30a-L6KD-PT-MtuΔI-CM-hPTH(1-34), pET30a-L6KD-PT-MtuΔI-CM m1-hPTH(1-34), pET30a-L6KD-PT-MtuΔI-CM m2-hPTH(1 -34), pET30a-L6KD-PT-MtuΔI-CM m3-hPTH(1-34)). Figure 1a Schematic diagram for expression and purification of teriparatide by cSAT method; Figure 1b It is a structural schematic diagram of the cSAT-based teriparatide fusion protein expression vector.

[0061] First construct the pET30a-L6KD-PT-MtuΔI-CM m2-hPTH(1-34) plasmid, the plasmid constructs the required primers, and the oligonucleotides shown in Table 1 are designed by oligo 7 and synthesized by Shanghai Sangong primers. First, the amino acid sequence of teriparatide hPTH (1-34) was obtained from the literature (Teriparatide: A Review). The seq...

Embodiment 2

[0067] Example 2: Expression and purification of teriparatide fusion proteins containing different mutant strains of intein MtuΔI-CM

[0068] Containing plasmids (pET30a-L6KD-PT-MtuΔI-CM-hPTH(1-34), pET30a-L6KD-PT-MtuΔI-CM m1-hPTH(1-34), pET30a-L6KD-PT -MtuΔI-CM m2-hPTH(1-34), pET30a-L6KD-PT-MtuΔI-CM m3-hPTH(1-34)) BL21(DE3) strains were inoculated into LB liquid containing 50 μg / mL kanamycin medium, and cultured in a shaker at 37°C until logarithmic phase (OD 600 =0.4~0.6), add final concentration 0.2mMIPTG, culture conditions are: 18°C, 250rpm, harvest cells after 24 hours, and measure bacterial concentration OD 600 (The following will be 1mL of OD 600 The amount of cells equal to 1 is called 1OD).

[0069] The thalline was treated with lysis buffer B1 (Tris of 2.4g, NaCl of 29.22g, NaCl of 0.37g 2 ·EDTA·2H 2 O was dissolved in 800mL water, adjusted to pH 8.5, added water to make up to 1L), resuspended to 20OD / mL, and subjected to ultrasonic crushing (crushing condition...

Embodiment 3

[0077] Example 3: Expression and purification of teriparatide hPTH(1-34) fusion protein using fermentation medium

[0078] Two BL21 (DE3) bacterial strains constructed in Example 1 (respectively containing pET30a-L6KD-PT-MtuΔI-CMm2-hPTH (1-34) plasmid and pET30a-L6KD-PT-MtuΔI-CMm3-hPTH (1 -34) plasmid) was inoculated into a fermentation medium (Shao-Yang Hu et al., 2004) containing 50 μg / mL kanamycin, and cultured in a shaker at 37°C until logarithmic phase (OD 600 = 0.4~0.6), adding a final concentration of 0.2mM IPTG, inducing at 18°C ​​for 24 hours, harvesting the cells, and measuring the bacterial concentration OD 600 . 1 mL of OD 600 A cell mass of 1 is called 1OD. The components of the fermentation medium used are shown in Table 4. Glucose was sterilized separately from other components, sterilized at 121°C for 20 minutes, and the trace element solution was filtered and sterilized on an ultra-clean bench with a 0.22 μm filter head. After the medium was prepared, kan...

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Abstract

The invention discloses a production and purification method of teriparatide hPTH (1-34), and belongs to the field of gene engineering. The method comprises the following steps of: sequentially connecting a gene sequence of an aggregation peptide, a gene sequence of a cutting tag and a gene sequence of human teriparatide hPTH (1-34) to form a gene of a fusion protein, and introducing the gene of the fusion protein into a host cell to obtain an engineering bacterium; culturing the engineering bacterium to express the fusion protein, then splitting the engineering bacterium, and performing centrifuging to take precipitate, so as to obtain an aggregate of the fusion protein; and cutting the aggregate of the fusion protein to obtain the human teriparatide hPTH (1-34). The yield and purity of the teriparatide hPTH (1-34) obtained through purification in the invention are very high; and the purification method is low in equipment requirement, few in steps, easy and convenient to operate, high in production efficiency and low in cost and can be applied to efficient preparation of the teriparatide hPTH (1-34).

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically, the invention relates to a production and purification method of teriparatide hPTH (1-34). Background technique [0002] Mature human parathyroid hormone (hPTH) is a polypeptide containing 84 amino acid residues secreted by the parathyroid gland. This hormone is a master regulator of the active form of calcium, phosphorus, and vitamin D in children's nerves, bones, gut, and blood (Murray et al., 2005). Studies have shown that the 34 amino acids at the N-terminal of parathyroid hormone (hPTH) have similar biological activities to intact parathyroid hormone (hPTH). This 34 amino acid peptide is teriparatide (hPTH, 1-34) (Cheng and Gupta, 2012). The relative molecular mass of teriparatide hPTH (1-34) is 4117.75, it is composed of 34 natural amino acids, and its sequence is: H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn -Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-...

Claims

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Application Information

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IPC IPC(8): C07K14/635C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/635C12N15/70C07K2319/00C07K2319/50
Inventor 林章凛任堂梅杨晓锋景艳芸
Owner SOUTH CHINA UNIV OF TECH
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