Method for secretory production of glycoprotein having human-type sugar chain using plant cell

a technology of plant cells and glycoproteins, which is applied in the direction of peptide sources, transferases, enzymology, etc., can solve the problems of tobacco plant antibody protein decomposition, unstable, and antibody protein produced within the cell is decomposed by the protease,

Inactive Publication Date: 2008-02-07
PHYTON HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The object of the present invention is to solve the above-described problems in conventional techniques and provide a method for the secretory production, in plant cells, of a glycoprotein which is stable, maintains its original physiological activity and is not an allergen, a plant cell capable of secreting such glycoprotein, and a glycoprotein having a human-type sugar chain secreted by this plant cell.

Problems solved by technology

However, the sugar chain structures of recombinant proteins actually produced within plant cells and purified have not been examined and these proteins are presumed to have a plant-type sugar chain structure.
Furthermore, in the case where another antibody molecule is produced from the same tobacco plant body, the antibody protein produced within the cell is decomposed by the protease and is unstable (see, L. H. Stevens, G. M. Stoopen, I. J. Elbert, J. W. Molthoff, H. A. Bakker, A. Lommen, D. Bosch and W. Jordi, “Effect of Climate Conditions and Plant Developmental Stage on the Stability of Antibodies Expressed in Transgenic Tobacco”, Plant Physiol., 124, 173-182 (2000)).
Because of this, the antibodies produced by tobacco plants are considered to be prone to decomposition by the protease.

Method used

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  • Method for secretory production of glycoprotein having human-type sugar chain using plant cell
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  • Method for secretory production of glycoprotein having human-type sugar chain using plant cell

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examples

[0091] The present invention is described below by referring to the Examples. The following Examples are only to illustrate but not to limit the present invention.

1. Cloning of Human β1-4 Galactose Transferase Gene

[0092] The β1-4 galactose transferase (hGt) (EC2.4.1.38) has been already cloned and a primary structure comprising 400 amino acids has been revealed (K. A. Masri et al., Biochem. Biophys. Res. Commun., 157, 657-663 (1988)).

(1) Primer Preparation and Template DNA

[0093] By referring to the report of Masri et al., the following primer were prepared.

(SEQ. ID NO: 1)hGT-5Eco:5′-AAAGAATTCGCGATGCCAGGCGCGCGTCCCT-3′(SEQ. ID. NO: 2)hGT-2Sal:3′-TCGATCGCAAAACCATGTGCAGCTGATG-5′(SEQ. ID. NO: 3)hGT-7Spe:3′-ACGGGACTCCTCAGGGGCGATGATCATAA-5′(SEQ. ID. NO: 4hGT6Spe:5′-AAGACTAGTGGGCCCCATGCTGATTGA-3′

[0094] As the template DNA, human genomic DNA, human placenta cDNA and human kidney cDNA purchased from Clontech were used.

(2) Cloning of hGT Gene cDNA

[0095] Using two combinations of (i)...

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Abstract

A method for the secretory production of a glycoprotein having a human-type sugar chain, comprising a step of introducing a gene of an enzyme capable of performing a transfer reaction of a galactose residue to a non-reducing terminal acetylglucosamine residue, and a gene of heterologous glycoprotein, to obtain a transformed plant cell, a step of culturing the plant cell, and a step of recovering the culture medium of the plant cell.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for the secretory production of a heterologous glycoprotein having a human-type sugar chain using a plant cell, a plant cell capable of secreting this glycoprotein, and a glycoprotein having a human-type sugar chain secreted by this plant cell. BACKGROUND ART [0002] Production of extraneous proteins using plant cultured cells is proceeding. For example, attempts are being made to produce the following proteins useful for humans using tobacco cultured cell: [0003] GM-CSF (see, E. A. James, C. Wang, Z. Wang, R. Reeves, J. H. Shin, N. S. Magnuson and J. M. Lee, “Production and Characterization of Biologically Active Human GM-CSF Secreted by Genetically Modified Plant Cells”, Protein Expr. Purif., 19, 131-138 (2000)), IL-2 and IL-4 (see, N. S. Magnuson, P. M. Linzmaier, R. Reeves, G. An, K. HayGlass and J. M. Lee, “Secretion of Biologically Active Human Interleukin-2 and Interleukin-4 from Genetically Modified Tobacco Cell...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H5/00C12N5/04C12P21/00C07K14/415C07K14/47C12N5/10C12N9/00C12N9/10C12N15/09C12N15/82C12P21/02
CPCC07K14/47C12N9/00C12N9/0065C12N15/8257C12P21/005C12N9/1051
Inventor FUJIYAMA, KAZUHITOSEKI, TATSUJI
Owner PHYTON HLDG
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