Production of high mannose proteins in plant culture

A high mannose and protein technology, applied in the field of plant culture and production of these proteins, can solve the problems of complex purification scheme, low biological activity, high cost of recombinant enzymes, etc.

Active Publication Date: 2006-12-06
普罗塔里克斯有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] A disadvantage of existing lysosomal enzyme replacement therapy treatments is that the in vivo biological activity of the enzymes is undesirably low, for example due to low uptake, reduced targeting of specific cellular lysosomes for accumulated substrates, and lysosomal Short half-life of in vivo action in enzymes
[0013] Another major disadvantage of existing GCD recombinases is their expense, which imposes a heavy economic burden on the health care system
The high cost of these recombinant enzymes is due to complex purification protocols and the considerable therapeutic doses required for current treatments

Method used

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  • Production of high mannose proteins in plant culture
  • Production of high mannose proteins in plant culture
  • Production of high mannose proteins in plant culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0216] Construction of expression plasmids

[0217] This example describes the construction of exemplary expression plasmids and is used in conjunction with the related examples below, as detailed below.

[0218] Use forward primer: 5′CA GAATTC GCCCGCCCCTGCA 3' (also represented by SEQ ID NO: 1) and reverse primer: 5' CTC AGATCT TGGCGATGCCACA 3' (also represented by SEQ ID NO: 2) amplifies the cDNA encoding hGCD (ATTC clone number 65696).

[0219] Purified PCR DNA products were digested with endonucleases EcoRI and BglII (see underlined recognition sequences in primers) and ligated into an intermediate vector with expression cassette CE-T digested with the same enzymes. CE-T includes the targeting signal MKTNLFLFLIFSLLLSLSSAEA (also represented by SEQ ID NO: 3) from the alkaline endochitinase gene [Arabidopsis], and the vacuolar targeting signal from tobacco chitinase A: DLLVDTM * (also represented by SEQ ID NO: 4).

[0220] The expression cassette was ...

Embodiment 2

[0224] Transformation of Carrot Cells and Selection of Transformed Cells Expressing rhGCD

[0225] This example describes an exemplary method of the invention for transforming carrot cells, as used in the following examples.

[0226] Carrot cells were transformed using the Agrobacterium transformation method described previously [Wurtele and Bulka (1989), supra]. The genetically modified carrot cells were plated on Murashige and Skoog (MS) agar medium containing antibiotics for selection of transformants. As shown in Figure 2, extracts prepared from calli were tested for expression of GCD by Western blot analysis using an anti-hGCD antibody and compared with recombinant glucocerebrosidase injection standard (positive control) and untransformed cells extract (negative control) for comparison. One callus (No. 22) was selected from various calli tested for expanded culture and protein purification.

[0227] Western blotting was performed as follows.

[0228] For this te...

Embodiment 3

[0238] Purification of recombinant active HGCD protein from transformed carrot cells

[0239] The recombinant hGCD expressed by transformed carrot cells was bound to the cell inner membrane but not secreted into the medium. Mechanical disruption of the cells left rGCD associated with insoluble membrane fragments (data not shown). The rGCD is then dissolved with mild detergent to separate it from cell debris and other insoluble components. The soluble enzyme was further purified using chromatographic techniques including cation exchange chromatography and hydrophobic interaction chromatography columns as described in the experimental procedures.

[0240]To separate medium from insoluble GCD, frozen cell pellets containing approximately 100 g wet weight cells were thawed and centrifuged at 17000 xg for 20 min at 4°C. Insoluble material and intact cells were washed by resuspending in 100 ml of wash buffer (20 mM sodium phosphate pH 7.2, 20 mM EDTA) and pelleted by cen...

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Abstract

A device, system and method for producing glycosylated proteins in plant culture, particularly proteins having a high mannose glycosylation, while targeting such proteins with an ER signal and / or by-passing the Golgi. The invention further relates to vectors and methods for expression and production of enzymatically active high mannose lysosomal enzymes using transgenic plant root, particularly carrot cells. More particularly, the invention relates to host cells, particularly transgenic suspended carrot cells, vectors and methods for high yield expression and production of biologically active high mannose Glucocerebrosidase (GCD). The invention further provides for compositions and methods for the treatment of lysosomal storage diseases.

Description

field of invention [0001] The present invention relates to transformed host cells for the production of high mannose proteins and, in particular, methods and systems for the production of these proteins by plant culture. Background of the invention [0002] Gaucher's disease is the most prevalent lysosomal storage disease. The disorder is caused by a recessive genetic disorder (chromosome 1 q21-q31) that results in a deficiency of glucocerebrosidase (also known as glucocerebrosidase), a membrane-bound lysosomal enzyme An enzyme that catalyzes the hydrolysis of the glycosphingolipid glucocerebroside (glucosylceramide, GlcCer) into glucose and ceramide. Gaucher disease is caused by point mutations in the hGCD (human glucocerebrosidase) gene (GBA), resulting in the accumulation of GlcCer in the lysosomes of macrophages. The characteristic storage cells, called Gaucher cells, are found in the liver, spleen, and bone marrow. Associated clinical signs include severe hepatosplen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C12N9/00C12N9/14C12N1/12C12N1/20C12N5/00C12N15/00C07H21/04A01H11/00C12M1/00C12NC12N9/24C12N15/82C12P21/00
CPCC12Y302/01045C12N9/2402C07K2319/04C12N15/8257C12N9/24C12P21/005C12N9/2465A61P25/02A61P3/00A61P31/22A61P43/00C12N15/00C12N15/09C12N15/11
Inventor Y·沙亚迪伊G·鲍姆D·巴特费德S·哈斯穆里A·勒科维茨
Owner 普罗塔里克斯有限公司
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