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Recombinant human growth hormone and its expression method in eukaryotic system

A technology of human growth hormone and nucleotide sequence, which is applied in the field of expression of recombinant human growth hormone and its eukaryotic system, can solve the problems of low expression, difficulty in industrialization, protein structure differences, etc., and achieve consistent biological activity Effect

Active Publication Date: 2022-04-05
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recombinant expression of foreign proteins in Escherichia coli generally adds a start codon to the N-terminus of the protein. Compared with the natural protein, there is an additional starting amino acid-methionine, which is easy to produce antibodies after the application of the growth hormone; another method is in the large intestine Secreted expression in Bacillus, but because Escherichia coli has no post-translational modification function, the structure of the obtained protein is somewhat different from the natural protein
There are also many studies on the expression of hGH in yeast systems, such as Shanghai Xinxinyuan (China Patent No.: CN101139583A), Wang Peng et al. (2008), Zhu Zhenhong et al. (2009) have successfully expressed hGH in yeast systems, but the expression level are low, it is difficult to achieve industrialization

Method used

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  • Recombinant human growth hormone and its expression method in eukaryotic system
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Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: Construction of different recombinant hGH expression vectors

[0037] 1. Construction of recombinant hGH expression vector containing Saccharomyces cerevisiae α-factor signal peptide and short spacer peptide

[0038] According to the hGH cDNA sequence published by GenBank (GenBank accession number: E00141), the inventor optimized the codon of the gene to obtain the gene of the present invention without hGH self-signal peptide, and the nucleotide sequence is shown in SEQ ID NO.5 , the amino acid sequence is shown in SEQ ID NO.6, and constructed into the pUC57 plasmid (provided by Nanjing GenScript Co., Ltd.), to obtain a long-term preservation plasmid, which is denoted as pUC57-hGH plasmid.

[0039] Using the pUC57-hGH plasmid as a template, different spacer short peptides EA were inserted behind the Xho I restriction site for PCR amplification. The primers used were as follows:

[0040] Recombinant hGH upstream amplification primer (SEQ ID NO.9) containin...

Embodiment 2

[0060] Example 2: Induced expression of different recombinant hGH engineering strains

[0061] 2.1 Screening of recombinant hGH host engineering strains:

[0062] The X-33 Pichia strain was prepared into electrocompetent cells according to the instructions of the Invitrogen Company's Easy SelectPichia Expression Kit. The six plasmids obtained in Example 1 were digested and linearized with Sac I restriction endonuclease (R0156S, purchased from New England Biolabs), and after ethanol precipitation, the linearized vector was electrotransformed into Pichia pastoris competent cells , spread on YPDS solid medium, and culture at 30°C until the transformants grow out.

[0063] Preparation of YPDS solid medium: Invitrogen Company Easy SelectPichia Expression Kit instructions provided, in which yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L, 182g / L.

[0064] 2.2 Methanol-induced expression of recombinant hGH engineering strains

[0065] Pick the host monoclonal en...

Embodiment 3

[0070] Embodiment 3: Purification of hGH recombinant protein

[0071] This patent mainly uses a two-step method to purify hGH protein. The first step uses cationic chromatography to enrich hGH and remove pigment, and the second step uses hydrophobic exchange chromatography to remove other foreign proteins. Specific steps are as follows:

[0072] 1. Pretreatment of fermentation broth

[0073] The shake flask supernatant obtained by the method in Example 2 was diluted with pH 4.5, 50 mM NaAc buffer solution to conductance <5 mS / cm, and filtered through a 0.45 μm filter membrane to obtain a purified sample after treatment, which can be purified by cationic chromatography.

[0074] 2. Purification by Cation Chromatography

[0075] Put the above treated purified sample on a 5ml Hitrap SP HP prepacked column, the equilibration buffer is 50mM NaAc, pH 4.5, the elution buffer is 50mM NaAc, 1.0M NaCl, pH 4.5, and wash according to 0-100% linear The hGH protein is mainly concentrat...

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Abstract

The invention relates to a recombinant human growth hormone and its eukaryotic system expression method, in particular to a recombinant human growth hormone coding gene sequence optimized for the expression system of Pichia pastoris, which is more conducive to the expression of hGH in Pichia pastoris Expression; at the same time, before the gene sequence, there are short spacer peptide sequences and signal peptide sequences specially designed for the expression of hGH in Pichia pastoris. In the present invention, the expression of hGH expressed in Pichia pastoris is significantly improved by optimizing the hGH gene, signal peptide and spacer short peptide, and the signal peptide and short peptide can be added to the carrier during design and use. It can also be added to the nucleotide sequence of the target protein; at the same time, the activity of the prepared hGH original protein has the same biological activity as that of commercially available products, so that the hGH of the present invention has an industrial application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering genes, and relates to a recombinant human growth hormone and a eukaryotic system expression method thereof. Background technique [0002] Human Growth Hormone (Human Growth Hormone, hereinafter abbreviated as hGH) is a protein hormone with a single peptide chain secreted by eosinophilic cells in the anterior pituitary gland. It is the most important hormone for human growth after birth, and has the ability to regulate human growth metabolism and other multiple functions. hGH contains 191 amino acid residues, the molecular weight is about 22kDa, and there is no glycosylation in the molecule. The molecule is connected by a pair of sulfur bonds between Cys53-Cys165 and Cys182-Cys189. These two disulfide bond pairs form the correct protein The conformation of the molecule is very important. hGH has a wide range of physiological functions, can affect almost all tissue types and cells, even including imm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/18C07K14/61C07K1/34C12N15/81
CPCC07K14/61C12N15/815
Inventor 马永范宇王安良
Owner ZONHON BIOPHARMA INST
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