Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-30
2002-08-06
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069700, C435S070100, C536S023720
Reexamination Certificate
active
06428960
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology and genetics. Specifically, the present invention relates to a method for producing recombinant baculovirus expressing a gene of interest by selecting for puromycin resistance that is conferred by that baculovirus.
BACKGROUND OF THE INVENTION
Baculoviridae comprise a family of viruses that infect insect cells, and can be genetically modified to serve as transfer and expression vectors for a highly productive recombinant protein expression system in the eukaryotic host. (Summers and Smith,
Tex. Agric. Exp. Stn. Bull
. B1555: 1-55 (1987); Davies, Bio/technology 12:47-50 (1994)). Exemplary baculoviruses include but are not limited to
Autographa californica
, Trichoplusia ni, Rachiplusia ou,
Galleria mellonella
and
Bombyx mori
. Of these,
Autographa californica is
probably the most fully characterized and widely used. The polyhedrin gene of baculoviruses is expressed at very high levels during the natural viral life cycle, but is dispensable in cell culture. This allows it to be replaced by almost any heterologous nucleotide sequence of interest. Any heterologous nucleotide of interest inserted so as to replace the polyhedrin gene may itself be controlled by the polyhedrin promoter, and thus expressed to similarly high levels.
Advantages of the baculovirus expression system include: (1) avirulence of baculoviruses in humans, (2) facility of protein expression in milligram quantities, and (3) ability of insect cells to grow at ambient temperature without the need for carbon dioxide (Kidd and Emery, 1993). Although both prokaryotic and eukaryotic cells have been used to express foreign genetic material, prokaryotic cells such as
Escherichia coli
are suitable for expressing foreign genetic material only if the gene product does not require post-translational modification such as glycosylation, phosphorlyation or signal peptide cleavage. Prokaryotic cells do not contain the machinery needed for such post-translational modification. Eukaryotic cells, such as insect cells which baculoviruses infect accomplish most eukaryotic post-translational modifications, including phosphorylation, N- and 0-linked glycosylation, acylation, disulphide cross-linking, oligomeric assembly and subcellular targeting which may be critical for the biological integrity of recombinant proteins (Davies, 1994). In contrast to the more commonly used bacterial expression systems, recombinant baculoviral expression generally produces soluble proteins without the need for induction or specific temperature conditions.
Manipulation of baculovirus genomes at the molecular level is challenging, as they comprise some 130 kb of DNA too large to be amenable to conventional plasmid cloning techniques. The traditional solution to this problem has been to introduce foreign genes by homologous recombination, as a cassette also comprising suitable promoter and termination sequences. This is accomplished by flanking the cassette, in a plasmid vector, by the viral DNA sequences which flank the point at which it is to be inserted. The efficiency of this process is usually less than 1%.
In the past, plaque purification has been used to proceed from this point to a clonal vector. Some traditional procedures are described by Smith et al., U.S. Pat. No. 4, 745,051, Smith et al, U.S. Pat. No. 4,879,236, Summers et al., U.S. Pat. No. 5, 169,784, Guarino et al., U.S. Pat. No. 5,077,214, Kang, U.S. Pat. No. 5,194,376, Matsuura et al, U.S. Pat. No. 5,229,293 and Murphy et al., U.S. Pat. No. 5,516,657, the disclosures of which are herein incorporated by reference. Recently, however, several different techniques have been developed to increase the frequency of recombination into the baculovirus genome. The most successful of these are described, infra.
The baculovirus genome has been reconstituted as a replicon which will propagate in the yeast
Saccharomyces cerevisia
. This was achieved by inserting a yeast Autonomously Replicating Sequence (ARS) into the polyhedrin locus of the baculovirus genome, along with a CEN (centromeric) sequence, which ensures stable low copy number segregation of the genome by acting as a mitotic centromere, and the URA3 selectable marker, to permit growth in a uracil-free medium. Thus the recombination of a polyhedrin promoter-driven foreign gene may be undertaken in yeast, and the resulting baculovirus genome extracted from the yeast cells and transfected directly into insect cells as a clonal virus. Advantages of this method include near 100% efficiency and the ability to manipulate in a foreign host genes whose products might be toxic to insect cells. However, a large number of manipulations are required making this procedure cumbersome and complicated.
The baculovirus genome has also been reconstituted as a replicon which may propagate in
Escherichia coli
. In a similar approach to that used in yeast, Luckow et al.,
J. Virology
, 67: 4566-4579, initially demonstrated that the baculovirus genome may be modified to replicate as a large plasmid in
Escherichia coli
, termed a ‘bacmid’. This may be performed by recombining a mini-F replicon into the polyhedrin locus, conferring autonomous replication and stable, low-copy number segregation of the genome, and the kanr selectable marker. The target site for the Tn7 bacterial transposon may also be introduced, as an in-frame insertion within the lacZa sequence from a pUC-based plasmid. This bacmid may therefore intra-allelically complement the defective &bgr;-galactosidase lacZDM15 of
Escherichia coli
hosts such as DH 1 OB. However,
Escherichia coli
DH l OB harboring a bacmid with a foreign gene inserted at this Tn7 site would remain lacZa−, enabling visual selection of colonies containing recombinant bacmids. The overall strategy of such a procedure is to accomplish the recombination and selection steps in the heterologous host, here
Escherichia coli
, and only then to transfer the finished product to insect cells. This strategy has similar advantages to the yeast system but also involves many steps.
Rather than reconstitute the baculovirus replicon in a heterologous host considered preferable for selection of recombinant species, an in vitro recombination reaction to transfer genes from transfer vectors to the polyhedrin locus of the virus has been developed. Exploiting the Cre recombinase of bacteriophage Pi and its substrate lovp, the gene transfer in this system is achieved by a single enzymatic crossover reaction. Both the target baculovirus genome (vaclox) and the transfer vector are engineered to contain lox sites. These 34 nucleotide sequences direct the Cre enzyme to convert the two substrate DNA molecules into topologically unlinked, recombinant products. The reaction proceeds stoichiometrically, with an efficiency of around 70%. This approach has the great advantage of simplicity, however, the maximum efficiency is only about 70%.
In 1990, Kitts et al.,
Nucl. Acids Res
. 18:5667-5672 (1990) derivatised wild-type baculovirus DNA, which exists as a covalently closed circular double-stranded molecule, by introducing a unique restriction site (Bsu361) at the polyhedrin locus. Linearising the baculovirus genome using this restriction site reduces the infectivity of the viral DNA on transfection into insect cells, however, cotransfection with a transfer vector driving recombination into the polyhedrin locus produces a three fold higher proportion of recombinant viruses.
In this strategy, the baculovirus containing the Bsu361 site was designated ACRP-SC (for single cut), and the two double crossover events transferring the foreign gene to be expressed, along with its own copy of the polyhedrin promoter and terminator sequences, are also present. About 10 to 25% of the progeny viruses from such a cotransfection are recombinant, but it should be noted that this is due to the reduced background of wild type viruses rather than an increase in the absolute number of recombinants.
A subsequent development of this system effectively combines it wit
Albala Joanna
Clark Robin
Franke Ken
McConnell Ian
Giotta Gregory
Onyx Pharmaceuticals Inc.
Park Hankyel T.
LandOfFree
Selection method for producing recombinant baculovirus does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Selection method for producing recombinant baculovirus, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Selection method for producing recombinant baculovirus will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2877395