Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-10-16
2002-11-26
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S348000
Reexamination Certificate
active
06485937
ABSTRACT:
FIELD OF THE INVENTION
Recombinant expression systems for the production of proteins, and particularly a system for rapidly generating recombinant silkworm baculoviruses.
Bombyx mori
nuclear polyhedrosis virus (BmNPV) with an efficiency approaching 100% has been developed.
BACKGROUND OF THE INVENTION
Since the advent of recombinant DNA technology, there has been steady growth in the number of systems available for the regulated expression of cloned genes in prokaryotic and eukaryotic cells. One eukaryotic system that has gained particularly widespread use is the baculovirus expression vector system, or BEVS, developed by Smith, G. E., and Summers, M. D., U.S. Pat. No 4,745,051).
The baculovirus expression vector system (BEVS) has now emerged as the preferred system for production of recombinant eukaryotic proteins in insect cells. Two major reasons for its wide spread popularity have been the high yields of recombinant proteins in a biologically functional form and minimal biohazard potential. BEVS employs strong viral promoters (polyhedrin or p10) for foreign gene expression in insect cells or larvae infected with a recombinant baculovirus. There are two types of baculovirus-based expression systems: the popular
Autographa californica
nuclear polyhedrosis virus (AcNPV) that infects Sf9/Sf21 cells and
Trichoplusia ni
larvae, and the
Bombyx mori
nuclear polyhedrosis virus (BmNPV) that infects silkworm cells (BmN) and larvae (
Bombyx mori
). Baculoviruses only infect insects and are further restricted by species-specific barriers, i.e., AcNPV will not infect BmN cells or silkworm larvae and BmNPV will not infect Sf9 cells.
The Family Baculoviridae have large, circular, double-stranded DNA genomes (at least 90-230 kilobases (Francki, R. I. B., et al., eds., in Archives of Virology, (1991), Supp. 2, pages 117-123. There are two Subfamilies, Nudibaculovirinae, which do not form occlusion bodies, and the Eubaculovirinae, are characterized by their ability to form occlusion bodies in the nuclei of infected insect cells. The structural properties of the occlusion bodies are used to further classify the members of this Subfamily into two genera: the nuclear polyhedrosis viruses (NPVs) and the granulosis viruses (GVs).
In nature, baculovirus-infected cells produce extremely high quantities of two major very late gene products polyhedrin (polh) and p10; which comprise 40-50% of the total cellular protein by the end of the infection cycle. Very late in infection (both in insects and tissue culture) a large proportion of the cellular transcriptional activity is dedicated to the polh and p10 promoters, which makes them ideally suited for driving high level expression of foreign genes that replace these non-essential viral genes. Yields up to 100 mg target protein per 10
9
cells or 50 silkworm larvae can be obtained. Viruses that lack the protective polyhedrin gene are innocuous in insects per Os (natural route of infection) but perfectly capable of establishing an infection when injected into the larvae manually. Hence, from an environmental, laboratory, manufacturing, and production point of view the silkworm larval system represents the safest system available to produce recombinant proteins and poses no threat to the sericulture industry.
The very late phase of baculovirus infection is distinct from the late phase of infection when budded viruses are formed. Consequently, expression of foreign proteins does not interfere with infectious virus production and virus replication. Target proteins can be directed to the appropriate subcellular location (including the cytoplasm, endoplasmic reticulum (ER), Golgi, plasma membrane, and nucleus) or secreted. Signal peptides of mammalian, plant, and yeast origin have been shown to direct proteins into the ER and to be properly cleaved in baculovirus-infected cells. Insect cells are capable of several post-translational modifications, which may be necessary to make some eukaryotic proteins functionally active. Myristoylation, phosphorylation, amidation, addition of fatty acids, sialylation, amino terminal and other eukaryotic protein modifications occur in baculovirus-infected cells. Glycosylation patterns are similar, but not identical, to those of mammalian cells. N-linked glycans (short or large mannose type) are added, as in mammals. Although, complex glycans are not formed, newer strains of engineered insect cells help overcome this deficiency. The silkworm larva-BmNPV based BEVS offers an additional advantage over Sf9-AcNPV based BEVS because of the possibility of expression in a variety of host cell types, thereby increasing the repertoire of post-translational modifications available for processing the recombinant proteins into their biologically functional form. Sf9 cells, being ovarian in origin, have a dedicated and limited capability of post-translational modifications in comparison to the silkworm larva.
The viral genome is very large (130 kb) and not amicable to direct manipulation, hence, the standard procedure for generating viral expression vectors has been to co-transfect insect cells with viral DNA and DNA of a transfer vector bearing the foreign gene under the control of the polhedrin promoter. Homologous recombination in vivo can replace a segment of the viral DNA by the modified sequence from the transfer vector, albeit at a very low frequency (0.1%-1%). Screening to identify a recombinant virus and separating it from parental virus can therefore involve considerable time and effort. Several modifications of this procedure that facilitate the identification of recombinant viruses by placing a reporter cassette adjacent to the gene to be expressed (Vialard et al., J. Virol. 1990; 64: 37-50; Vlak et al., Virol. 1990; 179: 312-320;Weyer et al., J. Gen. Virol. 1990; 71: 1525-1534 and Zuidema et al., J. Gen. Virol. 1990, 71: 2201-2209) or that increase the proportion of recombinant viruses (Kitts et al., Nucleic Acids Res. 1993; 18: 5667-5672; Peakman et al., Nucleic Acids Res. 1989; 13: 5403 and Kitts and Possee, Biotechniques 1993; 5:810-817) have been described. Recently, systems have also been developed for generating recombinant baculoviruses in yeast (Patel et al., Nucleic Acids Res. 1992; 20: 97-104.),
E. coli
(Luckow et al., J. Virol. 1993; 67: 4566-4579) or in vitro (Peakman et al., Nucleic Acids Res. 1992; 20: 495-500). A major bottleneck in the wide spread use of the BmNPV-based BEVS has been the tedious, time consuming plaque purification procedure required to isolate recombinant BmNPV expression vectors.
The major advantage of the BmNPV based expression system is that it can easily be expanded to an economical in vivo system using silkworm larvae. Because of its economic importance, the silkworm has been domesticated for thousand of years and techniques for mass rearing have been well-established. The silkworm larvae offers several additional advantages, e.g., it is easy to rear, it is easy to manipulate because of its large size, it has a relatively short life cycle (approximately 7 weeks), and its genetics and molecular biology have been well documented. The availability of artificial diets, availability of automated rearing equipment, and the fact that the larvae are non-allergenic to human handlers makes scale-up and mass production of recombinant proteins under sterile conditions very attractive in silkworm larvae. The silkworm BEVS has been applied for the production of useful biomolecules, such as pharmaceuticals, vaccines, enzymes, hormones, active viral insecticides, etc.
Foreign genes have been expressed using the AcNPV vector and their lepidopteran hosts, e.g.,
Trichoplusia ni
and
Heliothis virescens
. These species, however, are significantly smaller than
B. mori
, and often cannibalistic, so that special rearing conditions are required. Hence, for high yields and cost effective production of recombinant proteins in larval hosts the BmNPV-
B. mori
expression system is the best option.
However, a major bottleneck in the BmNPV-based BEVS has been the tedious, time consuming plaque purification
Palhan Vikas B.
Roeder Robert G.
Brown Stacy S.
Darby & Darby
Park Hankyel T.
The Rockefeller University
LandOfFree
System for rapid generation of recombinant baculovirus-based... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with System for rapid generation of recombinant baculovirus-based..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and System for rapid generation of recombinant baculovirus-based... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2937805