High efficiency peptide production in plant cells

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...

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 invention does not require the utilization of an infective viral agent; rather, non-infectious nucleic acid encoding a capsid-heterologous peptide fusion product is stably inserted into the genome of a plant cell, which can be cultured in a fermentation process to produce the peptide of interest. Such a process results in a less variable, and more stable host system for the expression of capsid fusion proteins containing heterologous peptides.

[0024] 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 recombinant capsid protein nucleic acid, and the expressed mutant capsid either cannot assemble into virus particles or contains a mutated target peptide. The present invention provides increased stability of the resultant heterologous peptide, with the additional benefits of precise control over growth conditions, batch to batch consistency, a high level of containment, and the ability to produce recombinant proteins in compliance with good manufacturing practices.

[0025] The capsid-fusion protein products can form virus like particles within the cell. The virus like particle may result in the improved efficiency of achieving a high purity of the recovered peptide. The virus like particles produced in the cell typically are not capable of infecting the plant cell. The viral capsid sequence can be derived from both trophic and non-trophic viruses, wherein trophism is determined by the specific plant cell utilized as the expression host. In one embodiment, the viral capsid protein is derived from a virus that exhibits a native or natural trophism towards the plant cell utilized to express the fusion product. In one embodiment, the viral capsid protein is derived from a virus that does not exhibit a native or natural trophism towards the plant cell utilized to express the fusion product. In one embodiment, the cell does not include artificially introduced viral proteins other than the desired capsid protein sequences utilized to produce the fusion product. In another embodiment, the cell includes artificially introduced viral proteins or nucleic acids other than the desired capsid protein sequences utilized to produce the fusion product, wherein the additional viral proteins or nucleic acids do not confer infectivity to the nucleic acid sequences. In one embodiment, the viral capsid is derived from a virus with a tropism to a different family of organisms than the plant cell expression host. In another embodiment, the viral capsid is derived from a virus with a tropism to a different genus of organisms than the plant cell expression host. In another embodiment, the viral capsid is derived from a virus with a tropism to a different species of organisms than the cell utilized to express the fusion product. In one embodiment of the present invention, the capsid is derived from a rod shaped plant virus. In a particular embodiment, the capsid is a rod shaped viral capsid derived from the group selected from Tobacco Mosaic Virus and Potato Virus X (PVX). In one embodiment of the present invention, the capsid protein is derived from an icosahedral virus. In a particular embodiment, the capsid is derived from a plant icosahedral virus. In a more particular embodiment, the icosahedral capsid is derived from the group selected from Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus, and Alfalfa Mosaic Virus.

[0026] The present invention also provides plant cells that include a non-infectious nucleic acid construct containing an expression cassette encoding for a fusion protein of a virus capsid and a recombinant peptide. The fusion peptide of the virus capsid and recombinant peptide is operably linked to a promoter and terminator that functions in plant cells. In one specific embodiment, the nucleic acid is genomically integrated in the plant cell, wherein the integration results in the stable inheritance and expression of the nucleic acid encoding the capsid fusion protein from generation to generation. In one specific embodiment of the present invention, the capsid protein is derived from an icosahedral virus. In one embodiment the cell produces virus like particles or soluble cage structures. In one embodiment, the plant cell is a Monocot or a Dicot. In a particular embodiment, the plant cell is Nicotiana tabacum. In an alternative embodiment, the plant cell is Oryza sativa. In one embodiment of the present invention, the recombinant peptide fused to the viral capsid protein is a therapeutic peptide useful for human or animal treatments. In one particular embodiment, the recombinant peptide is an antigenic peptide. In a particular embodiment, the antigenic peptide is a glycosylated antigenic peptide. In one embodiment, the plant cells or extracts containing capsid-recombinant peptide virus like particles containing an antigenic peptide can be administered as a vaccine to a human or animal. In an alternative embodiment, the purified capsid-recombinant peptide virus like particles containing an antigenic peptide can be administered as a vaccine to a human or animal. In one embodiment, the heterologous peptide is an antimicrobial peptide. In another particular embodiment, the recombinant peptide is a peptide that is toxic to the plant host cell when in free monomeric form. In one embodiment, the recombinant peptide fused to the capsid is at least 7, at least 8, at least, 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 75, at least 85, at least 95, at least 99, or at least 100 amino acids in length.

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 recombinant capsid protein nucleic acid, and the expressed mutant capsid either cannot assemble into virus particles or contains a mutated target peptide.

The virus like particles produced in the cell typically are not capable of infecting the plant cell.

Images