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Human augmenter of liver regeneration mutant, gene and application

A technology for enhancing factors and liver regeneration, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of protein folding and post-translational modification defects, poor rhALR activity, non-uniformity, etc., and achieve improved sulfhydryl oxidation activity and uniform product. , the effect of improving the protection effect

Active Publication Date: 2017-05-24
安徽新星生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the E. coli expression system is a prokaryotic expression system, there are defects in protein folding and post-translational modification when expressing eukaryotic proteins, and the finally expressed rhALR has poor activity.
However, the yield of rhALR expressed by Pichia pastoris is low and uneven, most of the protein is degraded, and the 7 amino acid residues at the N-terminus are cut off

Method used

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  • Human augmenter of liver regeneration mutant, gene and application
  • Human augmenter of liver regeneration mutant, gene and application
  • Human augmenter of liver regeneration mutant, gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Referring to the amino acid sequence (81-205 amino acids, a total of 125 amino acids) contained in CAB87993 (http: / / www.ncbi.nlm.nih.gov / protein / cab87993) in Genebank, the codon was optimized and entrusted by Sangon Bioengineering Shanghai The company synthesized the hALR gene (SEQ ID No.3) suitable for Pichia pastoris system expression, cloned it into pPICZα, and constructed the expression plasmid pPICZα-hALR; Anti-transformants, test tube expression screening to obtain expression strains.

[0037] BSM medium was used for fermentation, methanol induction was started when the OD reached above 6, and after 72-96 hours of induction, the tank was placed, and the supernatant was collected by centrifugation.

[0038]The rhALR expressed by yeast is chimeric with FAD molecules during the expression process, and the expression product is a natural dimer. The purified protein solution is transparent yellow, indicating that a stable protein-FAD complex has been formed during the ...

Embodiment 2

[0043] Pichia pastoris secretes and expresses recombinant protein outside the cell, which relies on Kex2 enzyme to recognize and cut KR ↓ (lysine-arginine) sequence. Therefore, when Pichia pastoris expresses the truncated hALR of 125aa, the expression product will be uneven, and most of them will be cleaved by the enzyme to cut off the 7 amino acids at the N-terminus. However, the study found that the deletion of the first 7 amino acids had no effect on its activity. Therefore, mutants missing the first 7 amino acids have been reported in the prior art.

[0044] Considering that the 71st and 72nd amino acid residues are also KR sequences, there may also be the possibility of being recognized and cut by the Kex2 enzyme in Pichia pastoris, so in the present invention, the 71st lysine is mutated into arginine (K71R), the 72nd arginine is mutated to lysine (R72K).

[0045] The natural hALR dimer is a homodimer formed by the reverse combination of two pairs of disulfide bonds at...

Embodiment 3

[0052] Oxidoreductase activity assay. According to the literature (Lisowsky T. et al, Digest Liver Dis., 2001, 33:173) method.

[0053] Accurately weigh glutathione (GSH) and prepare 10mM GSH mother solution with 20mM PB (pH7.5), and accurately weigh DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) to prepare with water into a 10 mM solution; yeast expressed purified rhALR, rhALR (Δ1-7) (prepared in Example 1) and rhALRs (prepared in Example 2), also diluted to 1 mg / mL with 20 mM PB (pH 7.5).

[0054] Prepare the reaction system according to Table 1.

[0055] Table 1 ALR sulfhydryl oxidase activity reaction assay

[0056]

[0057] Leave to react at 30°C for 6 hours, add 20 μL of DTNB to a final concentration of 20 μM, mix thoroughly, continue to react at 30°C for 5 minutes, and measure the absorbance at 412 nm. The sulfhydryl content is calculated according to the formula: sulfhydryl (μM)=OD 412 / 13.6.

[0058] Definition of ALR activity: reaction at 30° C. for 6 hours can r...

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Abstract

The invention discloses a human augmenter of liver regeneration mutant, a gene and application. The amino acid sequence of the human augmenter of liver regeneration mutant is shown as SEQ ID No. 1. According to the human augmenter of liver regeneration mutant, 7 amino acid residues at an N end are deleted and mutation of four single points (including C15S, K71R, R72K and C124S) is carried out to obtain a new mutant; a mutant protein obtained by expressing the gene of the mutant in pichia pastoris exists in a monomer form, and exposure of an active center (C62-C65) and FAD (Flavin Adenine Dinucleotide) combined nearby the active center is facilitated; an experiment result shows that the oxidization activity of sulfydryl is improved by 20 percent or more; the in vitro hepatocyte proliferation promoting capability and the protection effect on in vivo carbon tetrachloride live injuries are also improved; after a latent restriction enzyme site is mutated, obtained products are more uniform; guarantees are provided for large-scale operability of subsequent extraction and purification, and a reliable foundation is provided for development and clinical application.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a human liver regeneration enhancing factor mutant, gene and application. Background technique [0002] Various chronic liver diseases represented by HBV infection are difficult to be effectively and completely cured, leading to various liver fibrosis, liver damage, liver cirrhosis and liver cancer, etc., which have been a heavy burden on the health of our people for a long time. [0003] "Hepatopoietin" derived from a variety of animals is still used clinically; this type of drug has low purity, many impurities, unknown active ingredients, and severe clinical side effects. It is meaningful to seek a drug that can effectively promote liver regeneration. [0004] In 1994, Hagiya et al. (Hagiya M. et al., 1994, PNAS, 91:8142-8146) first discovered the rat ALR (augmenter of liver regeneration) gene. ALR in rats is a homodimer composed of two 125 amino acid polypeptide chains, ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19A61K38/44A61P1/16C12R1/84
CPCA61K38/00C12N9/0004C12N15/815C12N2800/102
Inventor 胡青松桂向东路遥石应辉李晓祥
Owner 安徽新星生物工程有限公司