Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-04
2001-05-01
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
06225060
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology and genetics. Specifically, the present invention relates to novel baculovirus expression systems and methods for expressing foreign genetic material by tranfecting the same into insect cells.
BACKGROUND OF THE INVENTION
A large amount of sequence information has been produced describing the genetic code of many smaller organisms. Tremendous accomplishments have been achieved in a far more expeditious manner than previously believed possible to similarly describe the genetic make-up of higher organisms such as humans. Undoubtedly, efforts will be intensified to accomplish sequencing the genetic code of these organisms, particularly those important to humans such as plants and other mammals. There now exists a particular need to develop rapid and accurate means for determining the functions of the particular genes and introns whose sequences are described by these sequence data in order to make use thereof. The first step in such an endeavor is expressing the gene products of sequenced genetic material. Critical technology involved in efficiently accomplishing this goal will likely utilize robotics capable of handling many gene or gene product samples simultaneously. Moreover, optimal methods for purifying expressed gene products will be utilized to further facilitate the process.
Baculoviruses infect insect cells. Both prokaryotic and eukaryotic cells have been employed to express foreign genetic material. However, prokaryotic cells such as
Eschericia 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. Hence, it is particularly important to develop novel and easily manageable recombinant expression systems in eukaryotic cells. The 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), and this has been exploited to produce functional recombinant protein from many diverse foreign genes.
Genetically modified baculoviruses are widely used to produce recombinant proteins. Davies, Bio/technology 12:47-50 (1994). Exemplary baculovirtises include but are not limited to
Autographa californica, Trichioplusia 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.
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 el 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 cercvisiae
. 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 el 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 1 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
Clark Robin
Davies Anthony
Giotta Gregory
ONYX Pharmaceuticals, Inc.
Park Hankyel T.
LandOfFree
Baculovirus expression system and method for high throughput... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Baculovirus expression system and method for high throughput..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Baculovirus expression system and method for high throughput... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2561536