Baculovirus enhancer-like sequence

Details Baculovirus enhancer-like sequence Baculovirus enhancer-like sequence

2002-07-09

2004-10-19

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

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

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

C435S320100, C435S325000, C435S348000, C536S023100, C536S024100, C536S023720

Reexamination Certificate

active

06806064

ABSTRACT:

BACKGROUND
The baculoviruses comprise a group of viruses that contain circular double-stranded DNA genomes of 90-160 kb (Blissard and Rohrmann, 1990; King and Possee, 1994; Miller and Dawes, 1979; and Smith and Summers, 1979). The circular 131-kb DNA genome of
Autographa californica
nuclear polyhedrosis virus (AcMNPV) is composed almost entirely of unique DNA sequences, except for several small repeated sequences known as homologous regions (hrs) that are interspersed within the viral genome (Ayers et al., 1994; Cochran and Faulkner, 1983; and Guarino et al., 1986). These hr sequences have been found to be enhancers for early gene transcription (Guarino et al., 1986; and Guarino and Summers, 1986) and as origins of DNA replication (Kool et al., 1993; Pearson, 1992).
The baculovirus expression vector system is one of the most popular systems for exogenous proteins production. Recombiannt proteins are expressed to very high levels due to the control of two very late polyhedrin and p10 promoters (Hasnain et al., 1997; Lopez-Ferber et al., 1995). However, the cell machinery critical for post-translational processing (e.g., glycosylation) is generally deteriorated during late and very late phases of baculovirus infection. Therefore, using early promoters for recombinant protein expression is an alternative approach to improve protein quality (Jarvis et al., 1990; and Jarvis et al., 1996). However, the activity of these early promoters is low compared with that of the very late promoters polyhedrin or p10.
SUMMARY
The invention is based, in part, on the discovery of an enhancer-like sequence upstream from the polyhedrin gene locus in the genome of baculovirus. This sequence, named herein “pu” was unexpectedly found to strongly activate the expression of a foreign gene early after viral infection, making pu very useful for the expression of recombinant proteins in insect cells with or without recombinant baculovirus. This pu sequence contains at least three open reading frames (ORFs) and can enhance the expression of full or minimal promoters. The ORFs contained in the pu sequence are ORF4 (SEQ ID NO:1), ORF5 (SEQ ID NO:2), and lef{acute over (
2
)} (SEQ ID NO:3). The pu sequence enhanced the expression of viral and cellular promoters to a much greater extent than the p10promoter. In addition, the pu sequence can act synergistically with the baculovirus hr sequence to enhance expression to surprisingly high levels.
Accordingly, in one aspect the invention features a nucleic acid construct, e.g., a plasmid construct or a viral construct (e.g., a baculovirus-based construct), useful for the high level expression of a heterologous protein in a cell, e.g., an insect cell. The construct includes a nucleotide sequence of less than 10,000 nucleotides, for example less than 5000 nucleotides, less than 2500 nucleotides, e.g., about 1500 nuclcotides, which sequence includes the sequences shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and can enhance the activity of an operably linked promoter. A non-limiting example of such a nucleotide sequence is the pu sequence described herein. It is understood that a limited number of additional nucleotides at the 5′ and/or 3′ end of the pu sequence would not interfere with the pu sequence's enhancer like activity. The enhancer like sequence is operably linked to a promoter, e.g., a cellular promoter, e.g., an insect cell promoter, or a viral promoter, which promoter is operably linked to a heterologous coding sequence in the constructs described herein. As the invention is not intended to encompass the enhancer-like sequence as it occurs in the naturally occurring baculovirus genome, the 5′ and/or 3′ sequence immediately flanking the nucleotide sequence that includes SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in the construct is different from the 5′ or 3′ sequence immediately flanking the sequence in a naturally occurring AcMNPV genome. In some embodiments, the constructs described herein also include a baculovirus hr sequence, e.g., the sequence shown as SEQ ID NO:4 or another hr sequence, e.g., as described in Ayres et al. (1994. Virology 202, 586-605). The hr sequence can operate cooperatively with the enhancer-like sequence described herein to produce unexpectedly high levels of expression from a promoter.
The constructs described herein can be used to express any heterologous coding sequence. As used herein, a “heterologous coding sequence” means that the coding sequence is not associated in nature with the promoter to which it is linked in the constructs of the invention. The heterologous coding sequence can be a non-viral coding sequence, e.g., an insect or mammalian coding sequence, e.g., a human coding sequence. Coding sequences that can usefully be used in the constructs described herein include, but are not limited to, sequences encoding enzymes, vaccines, antibodies, biologically active peptides, tumor antigens, or surface antigens. Nonlimiting examples of such sequences that can be expressed in a baculovirus system are described in, e.g., Huang et al. (2001) J Gen Virol 82:1767-76; Treanor et al. (2001) Vaccinel9:1732-7; Lieby et al. (2001) Blood 97:3820-8; Myles et al. (2001) Biochem J 357:225-32; tumor antigen: Soares et al.(2001) Protein Expr Purif 22:92-100; Fukumoto et al. (2001) J Clin Microbiol 39:2603-9.
The coding sequence is under the control of a viral or insect promoter, e.g., a minimal CMV promoter, a p35 promoter, a heat shock promoter, a p10 promoter, or a polyhedrin promoter, which promoters are known in the art. The promoter can be other than a polyhedrin promoter.
A construct described herein can also include one or more additional sequences necessary for expression of the heterologous coding sequence. For example, a construct described herein can include, e.g., a selectable marker, a 3′ untranslated sequence (UTS), a polyadenylation site, a foreign origin of replication e.g., a human origin of replication (described, e.g., in Burhans et al., 1994, Science 263: 639-640), or Epstein-Barr Virus replication origin and trans-acting factor. Such foreign origins of replication can increase gene expression in human cells. The constructs described herein can be made using standard molecular biology techniques, e.g., as described in Sambrook et al. Molecular Cloning: A Laboratory Manual, 3d ed., 2001, Cold Spring Harbor, which is hereby incorporated in its entirety.
In another aspect, the invention features a method of producing a polypeptide. The method includes: providing a nucleic acid construct described herein, e.g., a plasmid or viral construct described herein; introducing the nucleic acid construct into a cell, e.g., an insect cell; and allowing the cell to express a polypeptide encoded by the coding sequence.
In some embodiments, the nucleic acid construct is introduced into the cell by infection with a virus, e.g., a baculovirus, containing the nucleic acid construct.
In another embodiment, the nucleic acid construct is a plasmid construct. When the nucleic acid construct is a plasmid construct, the method can include the step of co-infecting the cell with a baculovirus.
In another aspect, the invention features a host cell, e.g., an insect cell, containing a nucleic acid construct described herein. Procedures for introducing constructs, e.g., baculovirus constructs, into cells, e.g., insect cells, are known in the art. See, e.g., Pfeifer et al., 1997, Gene 188:183-190; and Clem et al., 1994, J Virol 68:6759-6762.
A “nucleic acid construct” is defined herein as a nucleic acid molecule that has been modified to contain segments of nucleic acid that are combined and juxtaposed in a manner that would not otherwise exist in nature. The term encompasses plasmid and viral constructs.
The term “operably linked” is defined herein as a configuration in which a first sequence (generally a regulatory sequence) is placed at a position relative to a second sequence, e.g., a coding sequence or another control sequence, such that the first sequence affects the expression or activity of

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