High efficiency peptide production in plant cells

Active Publication Date: 2005-11-24
DOW AGROSCIENCES LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023] The present invention provides a process for the production of recombinant peptides, wherein non-infectious plasmids encoding fusion peptides comprising a viral capsid and a recombinant peptide of interest are stably inserted into the genome of a host plant cell and expressed in a suspension plant cell culture. The viral capsid-heterologous peptide fusion products can be expressed in vivo as virus like particles. The present inven

Problems solved by technology

It has been discovered that infectious viruses containing capsid-heterologous peptide fusion proteins utilized to express heterologous peptides of interest in whole plants exhibit genetic instability in the whole plant that results in mutations in the r

Method used

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  • High efficiency peptide production in plant cells
  • High efficiency peptide production in plant cells

Examples

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Effect test

example 1

Production of Antigenic Peptides in CCMV Virus Particles in Whole Cowpea Plants Inoculated with CCMV RNA1, RNA2, and Chimeric RNA3

[0211] Expression of Bacillus anthracis antigenic peptides was performed in whole plants, using cowpea chlorotic mottle virus (CCMV) with capsid proteins (CP) engineered to contain one of four different antigenic peptides.

[0212] DNA having the nucleotide sequence of CCMV RNA 1 and DNA having the nucleotide sequence of CCMV RNA 2 were each separately subcloned in the cloning vector pUC19 downstream from, and under the control of, a T7 promoter and upstream from a unique Xba I site. This produced plasmids pDOW2122 (CCMV RNA1) and pDOW2123 (CCMV RNA2).

[0213] DNA having the nucleotide sequence of CCMV RNA 3, engineered to contain five BamH I restriction enzyme cleavage sites, was further engineered for production of the recombinant capsid protein-encoding nucleic acid. Four DNA molecules, each encoding a different one of four exogenous peptides (four diffe...

example 2

Production of Antigenic Peptides in CCMV Virus Particles in Tobacco Suspension Culture Inoculated with CCMV RNA1, RNA2, and Chimeric RNA3

[0220] Expression of the same CCMV-peptide-encoding constructs was performed in plant cell suspension culture. Nicotiana tabacum NT1 cells were transfected by electroporation with RNA transcripts of CCMV RNA 3 coding for the chimeric CCMV coat proteins and CCMV RNA 1 and 2 coding for the replicase genes. Wild-type CCMV coat protein-encoding RNA 3 and engineered CCMV coat protein-encoding RNA 3 containing the appropriate BamHI restriction site but no inserts were used as controls.

[0221] 24 different RNA varieties were obtained by in vitro RNA transcription as described in the Example 1: one for RNA1, one for RNA2, 20 for chimeric RNA3, and two for RNA3 controls. In 22 different groups, two micrograms of each of three resulting RNAs (RNA1, RNA2, and one of the RNA3s) were transformed into tobacco cells by electroporation.

The Following Protocol wa...

example 3

Production of 4 Antigenic Peptides in CCMV Virus-like Particles in Tobacco Suspension Culture Transfected with Plant Expression Plasmids Encoding the Chimeric CCMV CPs

[0239] 1) Vector Construction:

[0240] Plasmid pIL-Tab358 was used as the plant expression vector. Restriction sites chosen for CCMV CP insertion were XbaI and EcoRI. This plasmid contains the Cassava Vein Mosaic Virus promoter upstream of the XbaI site and a Nos terminator downstream of the EcoRI site. The vector was prepared by digestion with XbaI and EcoRI and dephosphorylated before litigation with the inserts.

[0241] 2) Insert Construction:

[0242] CCMV CP-PA fusions were amplified by PCR out of pDOW2147 (PUC-CCMV-RNA3-CP129BamHI-PA1), pDOW2148 (pUC-CCMV-RNA3-CP129BamHI-PA2), pDOW2149 (pUC-CCMV-RNA3-CP 129BamHI-PA3), pDOW2150 (pUC-CCMV-RNA3-CP 129BamHI-PA4) using primers CCMV-CP-XbaI (SEQ ID NO:22) and CCMV-CP-EcoRI (SEQ ID NO: 23) to create pDOW2160 (pIL-Tab-CCMV129BamHI-PA1) (SEQ ID NO: 5), pDOW2161 (pIL-Tab-CCMV...

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Abstract

The present invention provides an improved process for the production of recombinant peptides. In particular, the present invention provides an improved process for the production of recombinant peptides in the form of viral capsid fusion proteins which can be assembled in vivo in plant cell suspension cultures. The invention also includes plasmids, sequences, and plant cells which allow for non-infectious viral capsid fusion peptide production.

Description

CROSS REFERENCE To RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional patent application Ser. No. 60 / 548,744, filed Feb. 27, 2004, entitled “High Efficiency Peptide Production in Plant Cells.”STATEMENT OF GOVERNMENT INTEREST [0002] This application is under a United States Government contract with the National Institutes of Health, National Institute of Allergy and Infectious Disease (NIAID), Cooperative Agreement No. 1-U01-AI054641-01.FIELD OF THE INVENTION [0003] The present invention provides an improved process for the production of recombinant peptides. In particular, the present invention provides an improved process for the production of recombinant peptides in the form of viral capsid fusion proteins which can be assembled in vivo in plant cell suspension cultures. The invention also includes plasmids, sequences, and plant cells which allow for non-infectious viral capsid fusion peptide production. BACKGROUND OF THE INVENTION [0004] Bacterial, y...

Claims

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Application Information

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IPC IPC(8): A61K39/00C07H21/04C07K14/08C12N5/04C12N15/82C12Q1/70
CPCA61K2039/5258A61K2039/6075A61K2039/64C07K14/005C12N2770/14023C12N15/8257C12N15/8258C12N2770/14022C07K2319/00A61P31/04A61P37/04
Inventor RASOCHOVA, LADADAO, PHILIP
Owner DOW AGROSCIENCES LLC
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