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Signal peptide for improving secretory expression of recombinant protein in mammalian cells and application thereof

A mammalian and cell technology, applied in mammalian proteins, animal cells, vertebrate cells, etc., can solve the problems of high cost and long screening time, and achieve the effect of reducing time and cost, reducing production cost and wide application prospects.

Active Publication Date: 2020-12-04
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the prior art, if you want to obtain a higher expression level, you need to screen multiple signal peptides individually for each secreted protein, and select a suitable signal peptide from them, which has the disadvantages of long screening time and high cost.

Method used

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  • Signal peptide for improving secretory expression of recombinant protein in mammalian cells and application thereof
  • Signal peptide for improving secretory expression of recombinant protein in mammalian cells and application thereof
  • Signal peptide for improving secretory expression of recombinant protein in mammalian cells and application thereof

Examples

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Effect test

Embodiment 1

[0036] In this example, 9 proteins were selected as verification proteins for the application effect of the signal peptide of the present invention. Under the same conditions, the signal peptide of the present invention and the signal peptide of the verification protein were used to compare the secretion and expression levels of the verification proteins in HEK293 cells. The specific operation is as follows:

[0037] 1. Construction of recombinant expression vector

[0038] First, construct a recombinant expression vector using the signal peptide of the present invention (SEQ ID No.1, see Table 1 for the specific sequence), named pHEK1.0: the amino acid sequence encoded by Shanghai Bioengineering Co., Ltd., such as SEQ ID No.21~SEQ The polynucleotide of ID No.29 (see Table 1 for the specific sequence), the sequence is SEQ ID No.39~SEQ ID No.47 (wherein the polynucleotide sequence encoding the signal peptide of the present invention is shown in SEQ ID No.39~SEQ ID No. The 1st ...

Embodiment 2

[0069] In this embodiment, Human CD73 protein is selected as the verification protein for the application effect of the signal peptide of the present invention in CHO cells. Under the same conditions, the signal peptide of the present invention and the signal peptide of the verification protein were used to compare and verify the secreted expression level of HumanCD73 protein in CHO cells. The specific operation is as follows:

[0070] 1. Construction of recombinant expression vector

[0071] First, a recombinant expression vector using the signal peptide of the present invention (SEQ ID No.1, see Table 1 for the specific sequence) is constructed: a polynuclear polynucleotide encoding the amino acid sequence SEQ ID No.29 (see Table 1 for the specific sequence) is synthesized by Shanghai Bioengineering Co., Ltd. Nucleotide, the sequence is SEQ ID No.47, a restriction endonuclease HindIII enzyme cutting site and a Kozak sequence are added at the 5' end; a restriction endonuclea...

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Abstract

The invention provides a signal peptide for improving secretory expression of recombinant protein in mammalian cells and application thereof. The invention firstly provides a polypeptide as shown in (a) or (b): (a) a polypeptide composed of an amino acid sequence shown in SEQ ID No. 1; and (b) a polypeptide which is obtained by substitution, deletion and / or addition of one or more amino acids to the amino acid sequence shown in SEQ ID No.1 and has the same function as the polypeptide composed of the amino acid sequence shown in SEQ ID No. 1. The polypeptide provided by the invention can be used as a signal peptide for secretory expression of target protein in mammalian cells, is beneficial for improving the secretory expression level of the target protein, screens out high-expression monoclonal cell strains, and greatly reduces the time and cost needed for screening high-expression signal peptides from secretory protein.

Description

technical field [0001] The present invention relates to a signal peptide and its application, more specifically, to a signal peptide which can be used to improve the secretion and expression of mammalian cell recombinant protein and its related application. Background technique [0002] Recombinant protein drugs are bright pearls in biological drugs. With the advancement of drug production technology and the increase in drug demand, the types of recombinant protein drugs in my country are also constantly enriched. [0003] Systems for the production of recombinant proteins using mammalian cells have been established for a long time. Compared with lower eukaryotic cell expression systems or prokaryotic expression systems, mammalian cell expression systems can guide the correct folding of proteins, provide complex N-type glycosylation and accurate O-type glycosylation and other post-translational processing modifications function, so the expression product is closest to natur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/85C12N15/62C12N5/10
CPCC07K7/08C07K14/4725C07K14/54C07K14/5418C07K14/70503C07K14/70521C07K14/70596C07K14/7155C07K2319/02C07K2319/21C12N5/0603C12N5/0682C12N5/0686C12N15/85C12N2510/02C12N2800/107
Inventor 秦丽丽范华李明月
Owner ACROBIOSYSTEMS INC
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