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Mammalian-type glycosylation in plants by expression of non-mammalian glycosyltransferases

a technology of plant glycosylation and non-mammalian glycosylation, which is applied in the field of transgenic plant glycosylation, can solve the problems of lack of agents infectious to humans, lack of glycoprotein production in plants, and lack of plant glycoprotein characteristics

Inactive Publication Date: 2012-02-09
STICHTING DIENST LANBOUWKUNDIG ONDERZOEK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Indeed, they are devoid of agents infectious to humans, and can accumulate the proteins of interest in storage tissues, such as seeds or tubers.
A drawback of glycoprotein production in plants relates to the glycosylation pattern of the glycoproteins produced in plants.
Moreover, plant glycoproteins lack the characteristic galactose-containing complex N-glycans found in mammals.
Such residues on glycoprotein are known to be immunogenic, which causes problems for certain applications of recombinant proteins carrying these sugars.

Method used

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  • Mammalian-type glycosylation in plants by expression of non-mammalian glycosyltransferases
  • Mammalian-type glycosylation in plants by expression of non-mammalian glycosyltransferases
  • Mammalian-type glycosylation in plants by expression of non-mammalian glycosyltransferases

Examples

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example 1

Identification of Putative Non-Mammalian β1,4 GalTs

[0124]The family of β-1,4-galactosyltransferases (GalT) comprises at least seven members, of which several have been cloned but only very few have been characterized. The β-1,4-galactosyltransferases of human and bovine origin have been best characterized and were shown to be able to add a galactose residue in β-1,4-linkage to terminal GlcNAc residue on an N-glycan. Putative GalT genes involved in N-glycosylation were identified among non-mammalian species gene sequences based on homology. FIG. 1 shows a Clustal W alignment of mammalian GalT gene sequences (human, mouse and bovine) and non-mammalian putative GalT gene sequences (chicken, zebrafish and frog). Remarkably, the mammalian amino terminus (boxed, solid lines), which is cytosolic and adjacent to the transmembrane region (TM, boxed, dashed lines) and which putatively is involved in Golgi-localization is not conserved in GalT orthologs from non-mammalian origin, such as, for ...

example 2

Cloning and Expression of Genes Encoding the Full-Length Chicken β1,4-GalT1 Enzyme and Variants Thereof.

[0128]Putative chicken β1,4-GalT1 (GGal; GenBank accession U19890; SEQ Ggal, SEQ ID NO:2) has been cloned earlier, although it was not shown to be capable of galactosylating N-glycans (Shaper et al., J Biol Chem 272, 31389-31399, 1997). In our lab, the cDNA fragment comprising residues 114 to 362 (SEQ GgGal114-362, SEQ ID NO:3) has been amplified from chicken spleen total RNA using RT-PCR with primers GgalLEEVAST and GgGaldw (see Table 1). The resulting fragment containing the C-terminus was digested with Xho I and Bam HI and then cloned into plasmid pCASeco, the latter being a pUC19 derivative in which the Hin dIII and Eco RI sites flanking the multiple cloning site have been used to insert the sequence SEQ CASeco at the same time removing these two sites and the Eco 31I-site in the backbone. This plasmid pCASeco was digested with Xho I and Bam HI to accommodate the C-terminal GG...

example 3

Cloning and Expression of Genes Encoding the Full-Length Zebrafish β1,4-GalT1 Enzyme and Variants Thereof.

[0136]A putative zebrafish β1,4-GalT1 (Machingo et al., Dev Biol 297, 471-82, 2006; NM—001017730) has been identified, but its function has not been proven and the inventors believe the identification to be incorrect. Starting from whole zebrafish total RNA, a full-length DGal gene (SEQ Dgal, SEQ ID NO:13) has been amplified using RT-PCR with primers DrGalup and DrGaldw (see Table 2). The resulting fragment was digested with Xba I and Bam HI and then cloned into likewise digested plasmid pCASeco (see above). Sequencing showed that it was virtually identical to unidentified Genbank accession NM—001077259, albeit with three mutations as shown here (start at +1 and non-silent mutations underlined): T126C, T230G, G862C. Amino acid sequence comparison of Dgal (SEQ Dgal, SEQ ID NO:14) with putative zebrafish β1,4-GalT1 as published (Machingo et al., Dev Biol 297, 471-82, 2006; NM—0010...

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Abstract

The present invention relates to non-mammalian β-1,4-galactosyltransferases that can be used in their wild-type or in modified forms. The invention further relates to transformed plants and plant cells expressing non-mammalian β-1,4-galacto-syltransferase and methods to produce glycoproteins with altered and preferably mammalian-type glycosylation. The invention additionally provides nucleic acid molecules and expression vectors of non-mammalian β-1,4-galactosyltransferases.

Description

FIELD OF THE INVENTION[0001]The invention relates to the field of glycoprotein processing in transgenic plants, in particular in such plants as used for the production of recombinant biopharmaceutical proteins.BACKGROUND OF THE INVENTION[0002]Recombinant protein production constitutes an important application of transgenic plants. In addition to the yield and the favorable cost of the field production of recombinant proteins, transgenic plants present certain advantages over other production systems, such as bacteria, yeasts, and animal cells. Indeed, they are devoid of agents infectious to humans, and can accumulate the proteins of interest in storage tissues, such as seeds or tubers. This facilitates their handling, their transportation and their storage at ambient temperature, while affording the possibility of subsequent extraction. Moreover, the transgenic plant, or some of its parts, can be utilized as vector of medicaments or of vaccines.[0003]Although the advantages of plant...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/82A01H5/10C07H21/00A01H5/06C12N5/10C12P21/00A01H5/00A01H5/12
CPCC07K2319/00C12N9/1051C12P21/005C12N15/8246C12N15/8257C12N15/8245C12N15/52C12N5/10A01H1/00
Inventor ROUWENDAL, GERARD JOHAN ADOLPHFLORACK, DIONISIUS ELISABETH ANTONIUSBOSCH, HENDRIK JAN
Owner STICHTING DIENST LANBOUWKUNDIG ONDERZOEK
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