Protein production in transgenic alfalfa plants

A transgenic alfalfa, transgenic technology, applied in the direction of anti-animal/human immunoglobulin, plant products, genetic engineering, etc., can solve problems such as infeasibility

Inactive Publication Date: 2001-04-04
AGRI & AGRI FOOD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such protocols use water and mechanical maceration and may not be feasible if proteolysis in the extract is severe, or if protease inhibitors are required during extraction

Method used

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  • Protein production in transgenic alfalfa plants
  • Protein production in transgenic alfalfa plants
  • Protein production in transgenic alfalfa plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] To exemplify the usefulness of alfalfa in expressing proteins, including multimeric proteins such as mAbs, a high affinity mAb (C5 -1). When tested with erythrocytes weakly sensitized with blood group antibodies, the C5-1 mAb gave reactivity similar to that of commercial rabbit polyclonal reagents. Integration and expression of transgene in alfalfa

[0090] The cDNAs encoding the C5-1 light and heavy chains were transferred into alfalfa plants following the protocol of Desgagnes et al. (1995). 15 plants and 25 plants of κ chain and γ chain were obtained respectively. The mRNA levels of kappa chain and gamma chain were monitored by RNA hybridization. The probe specifically hybridized to approximately 1.25 kbp kappa chain mRNA (Fig. 1A) and 1.75 kbp gamma chain mRNA (Fig. 1B). The alfalfa-derived mRNA was approximately 250 bp longer than the murine cDNA, indicating that in both cases the polyadenylation signal of gene 7 was used. Among the 29 F1 plants analyzed, 7 we...

Embodiment 3

[0094] Example 3 Produced in alfalfa and the stability of the transgenic protein extracted therefrom

[0095]Proteolysis of recombinant IgG has been shown to occur in Nicotiana tabacum and Arabidopsis thaliana (Hiatt et al., 1989; Ma et al. 1994). Although non-truncated IgG is synthesized in plants with a signal peptide that facilitates targeting to the inert extracellular matrix (De Wilde et al. 1996), it has been observed that IgG can be degraded by endogenous proteases upon extraction ( Hiatt et al. (1989), During et al. (1990), Ma et al. (1995), Ma and Hein (1995), Schouten (1996)). Since the present invention concerns the preparation of monomeric or multimeric proteins such as mAb C5-1 in alfalfa, the stability of such preparations was examined. 1) Protein stability in alfalfa extract compared to tobacco extract

[0096] To establish whether the protein was stable in crude extracts of alfalfa tissue, plant-derived or hybridoma-derived C5-1 were co-extracted in water in ...

Embodiment 5

[0111] The large-scale purification of embodiment 5 plant C5-1

[0112] 300 g of plant material were homogenized in 1.2 L of extraction buffer, pH 9.0, containing 50 mM borate and 4M NaCl. The homogenate was clarified by filtration on cheesecloth. The homogenate was then directly loaded by upflow onto an expanded bed of Streamline rProtein A matrix (Pharmacia). Samples were loaded at 4°C at a rate of 7ml / min. The column was washed with 250 ml extraction buffer and eluted with 50 mM sodium citrate, 50 mM sodium phosphate and 300 mM NaCl pH 3.0. The fraction (1.5 ml) was recovered and immediately neutralized with 100 µl of 1.5M Tris pH 8.8.

[0113] Selective expanded bed adsorption (STREAMLINE TM r Protein A), allowing the loading of partially clarified colloidal material-containing plant extracts at high flow rates. Using this technique, plant C5-1 was purified from non-clarified plant crude extracts, and the purified plant C5-1 was visualized as a single peptide by Coom...

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Abstract

This invention is directed to characterizing a host system suitable for the production of functional transgenic proteins, such as anti-human IgG, for use in applications requiring government regulatory approval. It is well known that regulatory agencies required stable, consistent master cell banks and master cell lines for the production of transgenic proteins in order to ensure sufficient material for appropriate characterization, clinical trials, and potential sales. Current plant production systems require the establishment of seed banks for this purpose. However, there are many draw backs related to such a system for the production of a continuous reliable transgenic protein source. An aspect of this invention is directed to characterizing a plant production system suitable for transgenic proteins that meet the stringent regulatory requirements. Another aspect of this invention exemplifies the production and characterization of an anti-human IgG for use as a blood grouping reagents, through the expression of corresponding genes in transgenic alfalfa plants. The cDNAs of the heavy and light chains of a human IgG-specific IgG2a(kappa) murine mAb (C5-1) were transferred into alfalfa through Agrobacterium infection. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained and plants from the Fl progeny (obtained by sexual crossing) were found to express fully assembled C5-1. Furthermore, the transgenic protein was stable in vivo, as well as during extraction and purification procedures. Purification yielded a unique H2L2 form with a reactivity indistinguishable from hybridoma-derived C5-1 in standardized serological tests. Results indicate that plant-derived transgenic proteins, such as mAbs can be used as diagnostic reagents as effectively as hybridoma-derived mAbs, and demonstrates the usefulness of the transformed alfalfa system to produce large amounts of proteins, including multimeric proteins such as mAbs.

Description

[0001] The present invention relates to the production of transgenic proteins in alfalfa plants. More specifically, the present invention relates to the production of multimeric proteins in transgenic alfalfa plants for various applications such as diagnostic assays. Background of the invention [0002] Full citations for references are listed at the end of the Examples section. [0003] The use of proteins for a variety of drug-related applications is subject to strict regulatory requirements established by the government. For example, in the United States, the Center for Biologics Evaluation and Research (CBER) has published a set of documents outlining requirements for the production and monitoring of transgenically produced proteins (see fttp:∥www.fda.gov / cberftp / html), at Canada has a similar set of regulations related to the use of biological products. The CBER document points out that biological products should be produced from reliable and sustainable resources to en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K16/18C12N15/09C12N15/13C12N15/82A01H5/00
CPCC07K14/70503C12N15/8258
Inventor L·P·维兹纳S·拉伯格R·巴津H·克豪迪R·莱米乌克斯G·阿拉德
Owner AGRI & AGRI FOOD
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