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A kind of preparation method of high-purity recombinant human brain natriuretic peptide

A technology of human brain natriuretic peptide and fusion protein is applied in the field of preparation of fusion tag protein and high-purity recombinant human brain natriuretic peptide. Expression, avoid renaturation, reduce the effect of production cost

Active Publication Date: 2020-04-21
四川泸州步长生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the fusion tag is not properly selected, soluble expression cannot be achieved, and the expression level is not high

Method used

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  • A kind of preparation method of high-purity recombinant human brain natriuretic peptide
  • A kind of preparation method of high-purity recombinant human brain natriuretic peptide
  • A kind of preparation method of high-purity recombinant human brain natriuretic peptide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of recombinant human brain natriuretic peptide fusion protein engineering bacteria

[0034] 1. Construction of pET30-a(+)-hBNP / BL21(DE3) engineering bacteria

[0035] The human brain natriuretic peptide mature protein sequence (GenBank accession number: NP_002512.1) is 32 amino acids, and the human brain natriuretic peptide gene sequence is shown in SEQ ID NO.1.

[0036] The tag protein is selected from the complete sequence of Escherichia coli MrsB (GenBank accession number: BAA15575.2); the amino acid sequence of the fusion tag protein is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.3 .

[0037] The street sequence was designed as -HHHHHHGGSDDDDK-, and the corresponding nucleic acid sequence was CACCATCATCATCATCATGGTGGTTCTGACGACGACGACAAG as a linker, and the C-terminal of the above-mentioned MrsB tag protein was connected to the N-terminal of the human brain natriuretic peptide mature protein sequence through the link...

Embodiment 2

[0043] Embodiment 2 Fermentation of recombinant human brain natriuretic peptide engineering bacteria

[0044] Streak LB plates (kan 100mg / L) with pET30-a(+)-hBNP / BL21(DE3) engineered bacteria and culture them in a constant temperature incubator at 37°C for about 16-18h until a single colony grows. A single colony of engineered bacteria was picked and inoculated into 20ml LB medium (kan 100mg / L), and cultured at 37°C and 230rpm for 8h. 0.1% was transferred to 250ml LB (kan 100mg / L), 1L conical flask, cultured at 37°C, 230rpm for 13h. Four bottles were cultured in parallel, 1000ml of bacterial solution was prepared, and 5% was inoculated into the fermenter NLF-2220L fermentation medium (TB medium). Before inoculation, the pH was adjusted to 7.0 with ammonia water, and the temperature during the fermentation process was controlled at 36°C. The pH value and dissolved oxygen of the fermentation medium are controlled by adding ammonia water and increasing the stirring speed and ven...

Embodiment 3

[0046] Example 3 Separation and Purification of Recombinant Human Brain Natriuretic Peptide

[0047] The bacteriostasis supernatant was loaded on Ni2+-Chelating Sepharose Fast Flow (GE Healthcare) chelation chromatography medium treated with 0.2M NiSO4 and equilibrated with 20mM Tris-HCl (pH8.0), and 200mM imidazole (containing 20mM Tris- HCl, pH 8.0) elution; the purpose fusion protein collected was desalted, and 0.5U enterokinase (50mM Tris-HCl, 2mM CaCl , 0.1% Tween-20, pH 8.0) was added to each 1mg fusion protein at 6- Digest the fusion protein for about 17 hours at 8°C.

[0048] The digested target protein is loaded on Ni2+-Chelating Sepharose Fast Flow, and the target protein and fusion tag are simultaneously hung on the column. However, the binding capacity of the target protein on the column was low, and the target protein was eluted with 50 mM concentration of imidazole (Shanghai Sangong, batch number: B421BA0025), and the peak of the eluted protein was collected. A...

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Abstract

The invention provides a method for preparing high purity recombinant human brain natriuretic peptides, which comprises steps: encoding genes of mature brain natriuretic peptides are optimized, and a fusion partner which is easy to express in high efficiency in escherichia coli is fused on an N end of the mature brain natriuretic peptide which is encoded through a joint. The invention further provides an expression vector comprising the nucleic acids of encoded fusion proteins, engineering bacteria comprising the expression vector and a method for preparing recombinant human brain natriuretic peptides by utilizing the engineering bacteria. The recombinant human brain natriuretic peptides which are prepared through the method for preparing the high purity recombinant human brain natriuretic peptides have the advantages of same biological activity with native proteins, high purity and low cost and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a preparation method of high-purity recombinant human brain natriuretic peptide. The invention further relates to a fusion tag protein for preparing recombinant human brain natriuretic peptide which is easy to express efficiently. Background technique [0002] Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide (BNP), is another member of the natriuretic peptide system after atrial natriuretic peptide (ANP). It was found in the pig brain, and subsequent studies found that brain natriuretic peptide is mainly synthesized and secreted in the ventricular myocardium, which can promote sodium excretion, urination, and vasodilation, and has a strong anti-proliferation effect on vascular smooth muscle cells and endothelial cells Antagonizing the vasoconstriction of the renin-angiotensin-aldosterone system (RAAS), it plays an important role in the balan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/245C07K19/00C07K14/58C12N15/70C12N1/21
CPCC07K14/245C07K14/58C07K2319/21C07K2319/23
Inventor 李树刚张伟辛渝龚会英但国平柴新娟王勇刘新于廷和
Owner 四川泸州步长生物制药有限公司
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