Method of producing a chimeric protein

Details Method of producing a chimeric protein Method of producing a chimeric protein

1997-04-18

2001-05-15

Allen, Marianne P. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S069700, C435S068100, C435S235100, C435S239000, C435S320100, C435S948000

Reexamination Certificate

active

06232099

ABSTRACT:

This invention relates to a method of producing a chimeric protein, eg a biologically active protein such as an antibiotic peptide.
Typical antibiotic peptides include the marginins, 23 amino acid-long alpha-helical peptides, originally identified from frog skin, which have a significant antibacterial activity; the defensins which combat bacteria, fungi and some enveloped viruses such as herpes simplex virus and HIV; and the protegrins which are 16-18 amino acid-long antibiotic peptides with strong biocidal activity.
The protegrins form part of an array of antibiotic peptides that are used by mammalian phagocytes to destroy invading pathogens through non-oxidative processes. Typically the protegrins include 4 cysteine residues and form a double-stranded &bgr;-sheet structure and show sequence similarity with the antibiotic defensin peptides that are also involved in phagocyte defence responses. The defensins are cationic, cysteine-rich peptides of 29 to 34 amino acids that are formed almost entirely of &bgr;-sheet structures and that have been shown to have biocidal activity against bacteria, fungi and some enveloped viruses, including herpes simple virus and HIV. Both the protegrins and defensins are expressed in phagocytes as pre-pro-proteins which are cleaved to release the biocidal peptides from the carboxy-terminus of the protein.
Because of their antibacterial activity it may not be convenient to synthesize these antibiotic peptides by genetic engineering in conventional prokaryotic expression systems. Solution synthesis of large amounts of these peptides with a variety of amino acid modifications may be possible, but is not currently considered commercially viable, since a significant drop in yield occurs in the manufacture of peptides of over 25-30 amino acid residues.
Eukaryotic expression systems (yeast, insect, animal or plant cells which produce foreign proteins or peptides) may be necessary if there is a need for post-transitional modification of the desired protein, but fermentation processes for such eukaryotic expression systems are expensive to maintain, provide little flexibility in terms of scaling the process up to industrial production levels and are very susceptible to contamination. Processing and purification of the desired protein can also be complex and costly.
The use of plants and benign plant viruses offers an opportunity to produce foreign proteins with minimal host cell contamination, thereby reducing contamination problems which could affect successful achievement of the required regulatory body approval for human or veterinary applications.
It has been proposed in WO92/18618 to use plant viruses as vector systems for the expression of foreign nucleotide sequences. WO92/18618 describes the use of a Comovirus (Cowpea Mosaic Virus or CPMV) as an effective vector for such expression and also mentions other spheroidal viruses such as HIV and Picorna-viruses. Picornaviridae generally comprise particles of 22-30 nm having cubic symmetry; Comoviridae have a pair of 28 nm particles with a similar symmetry, and HIV is a member of the Retroviridae which are generally enveloped 100 nm particles containing an icosahedral nucleocapsid.
One disadvantage of the system disclosed in WO92/18618 is that the geometry of the spheroidal viruses precludes large proteins from being produced, since the size and number of chimeric proteins per virus particle (generally 60 for icosahedral virus particles) is limited by the spheroidal geometry of the virus.
Construction of chimeric proteins in such viruses is also limited to the insertion of the foreign component into a loop in a native virus protein, eg the &bgr;-B to &bgr;-C loop in VP23 of CPMV, where such insertion does not affect the geometry of the coat protein and/or its ability to self-assemble into a virus particle (virion). As can be appreciated, the size of the peptide which can be tolerated in such an insertion is fairly limited; polypeptides of a maximum of 26 amino acids in length are cited by WO92/18618. Larger polypeptides present in internal insertion sites in coat or capsid proteins of the viruses exemplified may result in disruption of the geometry of the protein and/or its ability to successfully interact with other coat proteins leading to failure of the chimeric virus to assemble. Modified viruses which cannot self-assemble might not infect other host cells and produce whole plant infection. This possible lack of ability to spread the infection of the modified virus constitutes a significant disadvantage in the prior system.
The present invention contemplates the use of benign high copy number rod-shaped viruses, preferably plant viruses such as potato virus X (PVX), to produce foreign protein connected to viral coat protein subunits. When assembled, the virus particles comprise long helical arrays of more than 1000 identical chimeric proteins (which are typically coat protein—foreign protein fusion molecules) per virion. Generally the foreign protein portion will be displayed on the outer surface of the virus particles.
A suitable proteolytic degradation site (eg elastase or CNBr) may be engineered into the chimeric protein to permit release of the foreign protein portion from purified virus material. Given the size of the foreign protein and the relevant composition of the possible viruses, it is estimated that between 10 and 30% of the total weight yield of virus particle could comprise the foreign protein. Release of the foreign protein by proteolytic cleavage can be a simple purification regime, followed by removal of the residual innocuous plant virus itself. Yields of plant virus up to 5 g per kg wet weight of leaf from potato or tobacco are possible and hence the yields of foreign protein could be very substantial.
If the foreign protein is left attached to the chimeric protein in the virus particle, the whole virus particle can also be used as a vector for expression and presentation of peptide epitopes for vaccination of animals and/or the delivery of therapeutic single-stranded RNA molecules. This may be utility in the delivery of anti-sense or triplex nucleotides.
The present invention provides a method of producing a chimeric protein comprising:
a. providing a rod-shaped recombinent virus or pseudovirus containing a polynucleotide encoding a chimeric protein having a first (viral) portion and a second (non-viral) portion, the chimeric protein being capable of assembly into a virus particle such that the second portion is disposed on the exterior surface of the assembled virus particle;
b. infecting a host cell with the virus or pseudovirus; and
c. allowing replication of the virus or pseudovirus and expression of the chimeric protein in the host cell.
The term “rod-shaped” as applied herein to viruses includes filamentous or flexuous viruses, which are preferred. It is advantageous to use a virus which is flexuous (ie which can bend easily) since chimeric proteins with large second portions may be able to assemble more easily into virus particles (virions) which are flexuous than those which are rigid. PVX is preferred since it forms a flexuous virion.
The virus or pseudovirus can preferably assemble in the host cell to produce infective virus particles which comprise nucleic acid and chimeric protein. This enables the infection of adjacent cells by the infective virus or pseudovirus particle and expression of the chimeric protein therein.
The host cell can be infected initially with virus or pseudovirus in particle form (ie in assembled rods comprising nucleic acid and protein) or alternatively in nucleic acid form (ie RNA such as viral RNA; cDNA or run-off transcripts prepared from cDNA) provided that the virus nucleic acid used for initial infection can replicate and cause production of whole virus particles having the chimeric protein.
The term “pseudovirus” as used herein means a virus-derived nucleic acid sequence optionally assembled into particles and having an incomplete viral genome as compared to wild-type virus but retaining sufficient viral genes to allow replication and ass

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