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Production and purification method and application of disulfide bond-containing polypeptide human brain natriuretic peptide hBNP

A technology of human brain natriuretic peptide and purification method is applied in the field of production and purification of disulfide bond-containing polypeptide human brain natriuretic peptide hBNP, which can solve the problem that there is no efficient expression of disulfide bond-containing polypeptide and human brain natriuretic peptide. The problems of high price of BNP products, cumbersome purification steps, etc., achieve the effect of being conducive to commercialization, simple purification, and simplified process

Pending Publication Date: 2022-04-15
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These technical processes require cumbersome purification steps, and require the use of various column chromatography techniques, such as affinity chromatography, gel exclusion chromatography, etc., with low yield and high cost, resulting in the loss of human brain natriuretic peptide BNP high product price
In particular, there is still no efficient method for expressing disulfide bond-containing polypeptides

Method used

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  • Production and purification method and application of disulfide bond-containing polypeptide human brain natriuretic peptide hBNP
  • Production and purification method and application of disulfide bond-containing polypeptide human brain natriuretic peptide hBNP
  • Production and purification method and application of disulfide bond-containing polypeptide human brain natriuretic peptide hBNP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Construction of human brain natriuretic peptide hBNP fusion protein expression vector containing intein MtuΔI-CM

[0071]The expression vectors used in the examples of this application pET30-L6KD-MtuΔI-CM-BNP, pET30-L6KD-MtuΔI-CM m1-BNP, pET30-L6KD-MtuΔI-CM m2-BNP, pET30-L6KD-MtuΔI-CM m3 The construction process of -BNP and pET30-α3-MtuΔI-CM-BNP is similar. The following takes the construction of pET30-L6KD-MtuΔI-CM-BNP as an example. The required primers are designed by oligo 6 and synthesized by Shanghai Sangong as shown in the table Oligonucleotide primers indicated in 1.

[0072] Table 1 Oligonucleotide primers used in this embodiment

[0073]

[0074]

[0075] Use Y200630-BNP-1F, Y200630-BNP-2R, Y200630-BNP-3F, Y200630-BNP-4R, Y200630-BNP-5F, Y200630-BNP-6R, Y200630-BNP-7F, Y200630-BNP-8R primers as The template was annealed and spliced ​​by PCR reaction (PCR instrument (Bio-rad / C1000 Touch)) to obtain brain natriuretic peptide BNP polynucleotide...

Embodiment 2

[0080] Example 2: Expression and purification of human brain natriuretic peptide hBNP fusion protein in LB medium

[0081] The strain constructed in Example 1 (containing each plasmid as described above) was inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultivated in a shaker at 37° C. to logarithmic phase (OD 600 =0.4-0.6), add a final concentration of 0.2mMIPTG, induce at 18°C ​​for 24 hours, harvest the cells, and measure the bacterial concentration OD 600 . 1 mL of OD 600 A cell mass of 1 is called 1OD.

[0082] 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 800 mL of water, adjusted to pH 8.5, added water to make up to 1 L), resuspended to 50 OD / mL, and subjected to ultrasonic crushing (crushing conditions: power 200 W, ultrasonic time 3 sec, interval time 3 sec, ultrasonic frequency 99 times). Centrifuge at 4°C and 15000g for 20min, and collect the supernatant and precip...

Embodiment 3

[0088] Example 3: Expression and purification of human brain natriuretic peptide hBNP fusion protein in fermentation medium

[0089] The strain constructed in Example 2 was inoculated into a fermentation medium containing 50 μg / mL kanamycin (Shao-YangHu et al., 2004), and cultivated in a shaker at 37° C. to logarithmic phase (OD 600 =0.6-0.8), 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 3.

[0090] Table 3 Fermentation Medium Components

[0091]

[0092] 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, kanamycin with a final concentration of 50 mg / L was added before use.

...

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Abstract

The invention relates to a production and purification method of disulfide bond-containing polypeptide human brain natriuretic peptide rhBNP. The method comprises the following steps: sequentially connecting a gene sequence of an aggregation peptide, a gene sequence of a cleavage tag and a gene sequence of human brain natriuretic peptide rhBNP 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 engineering bacteria to express the fusion protein, then splitting the engineering bacteria, and 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 brain natriuretic peptide rhBNP. The yield and purity of the human brain natriuretic peptide rhBNP obtained through purification are very high, and especially after self-cutting and centrifugal operation, the purity of 99% or above can be achieved only through two-step standardized column chromatography. The purification method has the advantages of low requirements on equipment, few steps, simplicity and convenience in operation, high production efficiency and low cost, and can be applied to large-scale efficient biological preparation of the human brain natriuretic peptide rhBNP.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically, the invention relates to a method for producing and purifying a disulfide bond-containing polypeptide human brain natriuretic peptide hBNP. Background technique [0002] Since FDA approved recombinant insulin for diabetes treatment in 1982, more and more proteins / peptides have been approved as therapeutic drugs in EU and US (Agyei D et al., 2017). Peptide drugs have attracted much attention in recent years because of their high specificity, good curative effect, and small toxic and side effects. Kits and other aspects (Leader et al., 2008). In the 1980s, almost all peptides entering clinical development were less than 10 amino acids in length. In the ensuing decade, thanks to improvements in peptide synthesis and production technology, the average peptide length of peptide drugs entering clinical development has increased. In the past decade, the proportion of peptide ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C07K14/58C07K1/18C07K1/22A61K38/22A61P9/04
Inventor 杨晓锋杨莎林章凛景艳芸
Owner SOUTH CHINA UNIV OF TECH
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