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Expression and purification method of anti-cancer and anti-inflammatory polypeptide lunasin in mammalian cell cho-s

A mammalian and cell-based technology, applied in the field of protein engineering, can solve the problems of late development of mammalian cell expression system, high cost of cell culture, and difficult conditions to master, and achieve the effects of low cost, convenient and simple protein purification, and reduced release

Inactive Publication Date: 2020-03-31
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI +1
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Problems solved by technology

Compared with the prokaryotic expression system of Escherichia coli and the eukaryotic expression system of Pichia pastoris, the mammalian cell expression system was developed late, and the cost of cell culture is high, the conditions are difficult to master, and it is easy to pollute, which also affects its wide application to a certain extent.
Therefore, there is no report on the recombinant expression of Lunasin polypeptide in mammalian cells

Method used

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  • Expression and purification method of anti-cancer and anti-inflammatory polypeptide lunasin in mammalian cell cho-s
  • Expression and purification method of anti-cancer and anti-inflammatory polypeptide lunasin in mammalian cell cho-s
  • Expression and purification method of anti-cancer and anti-inflammatory polypeptide lunasin in mammalian cell cho-s

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Embodiment Construction

[0038] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0039] 1) Search the Lunasin gene sequence in the NCBI database, and chemically synthesize the Lunasin gene in vitro. The sequence of Lunasin gene is shown in SEQ ID NO.1.

[0040] 2) Design primers for amplifying Lunasin gene. Forward primer (Lunasin-F) is CG TCCAAATGGCAGCACCAGC (SEQ ID NO.2), the restriction site is EcoRI (italics); the reverse primer (Lunasin-R) is CCG GTCGTCGTCATCATCATCATCGTC (SEQ ID NO. 3), the restriction site is XhoI (italics).

[0041] 3) PCR amplification of Lunasin gene. The amplification conditions were pre-denaturation at 95°C for 3 minutes, 30 cycles at 95°C for 30s, 55°C for 30s, and 72°C for 50s, and extension at 72°C for 10 minutes. The results of agarose gel electrophoresis showed that the PCR fragment conformed to the expected size of the target gene, see figure 1 .

[0042] PCR amplification system...

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Abstract

The invention discloses expression and purification methods of anti-cancer and anti-inflammatory polypeptide Lunasin in mammalian cells CHO-S. The expression and purification methods of Lunasin polypeptide in mammalian cells comprise the following steps: (1) carrying out in-vitro chemical synthesis of a Lunasin gene; (2) carrying out PCR amplification on the Lunasin gene; (3) transforming a eukaryotic expression vector; (4) connecting the Lunasin gene to the transformed expression vector pZWCT1 of the mammalian cells; (5) identifying positive clone; (6) carrying out transient transfection on the mammalian cells CHO-S by adopting expression vector plasmids; (7) producing Lunasin polypeptide through high-density cell culture; and (8) carrying out separation and purification on the Lunasin polypeptide. With the expression and purification methods, the large-scale production of the Lunasin polypeptide becomes possible, the cost is low, the yield is high, the protein modification is accurate, and meanwhile, the protein purification is convenient and simple.

Description

technical field [0001] The invention belongs to the field of protein engineering, and relates to a method for expressing and purifying anti-cancer and anti-inflammatory polypeptide Lunasin in mammalian cell CHO-S. Background technique [0002] Lunasin polypeptide is an aspartic acid (Asp)-rich active peptide (molecular weight 5.5KD) that was first isolated from soybean. It consists of 43 amino acids and its sequence is SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDDDDDDD. The function of the 1-22 amino acid sequence at the N-terminal is still unclear, the 23-31 amino acid sequence can make Lunasin localize to the chromosome histones H3 and H4, and the 32-34 amino acid Arg-Gly-Asp (RGD) is A cell adhesion motif that allows Lunasin to enter the nucleus. Its C-terminus has 9 Asp in a row to make the Lunasin polypeptide bind to the centromere of the chromosome, so that the microtubules cannot adhere to the centromere (Hernández-Ledesma et al.2009). The above properties of Lunasin poly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/85C12N5/10C07K14/415C07K1/22
Inventor 薛佳宇周广灿王元涛张艳梅
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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