Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-11-29
2002-10-08
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S070100, C435S325000, C435S348000
Reexamination Certificate
active
06461863
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of recombinant DNA vectors, and particularly concerns vectors useful for producing a desired protein of interest in an insect cell that has modifications similar to the same protein produced in mammalian cells. More particularly, it concerns recombinant baculovirus vectors that are used to infect or stably transform insect cells, directing the production of oligosaccharide processing enzymes, other protein modification enzymes and proteins which aid in proper protein folding, thereby obtaining the desired protein. The invention also concerns insect cells with stably integrated protein modification enzymes, and methods utilizing the vectors, viruses and cells disclosed herein.
2. Description of Related Art
One of the major benefits provided by recombinant DNA technology is the ability to express cloned genes in a heterologous host, which facilitates the isolation of large amounts of foreign gene products for further study or direct practical applications. Bacterial systems typically provide high expression levels, but lack eucaryotic protein processing capabilities. Many biomedically important proteins are processed, and the lack of processing can alter their folding, transport, stability and/or function (Welply, 1991). Also, the foreign gene product is often deposited as an insoluble inclusion body. Mammalian systems can provide protein processing, but expression levels are much lower and mammalian cells are much more expensive to cultivate. An ideal system would combine high expression levels, proper protein processing, and still be relatively inexpensive.
Baculoviruses are DNA-containing viruses that infect insects or other invertebrates (Adams and McClintock 1991). Baculovirus vectors usually provide high levels of foreign gene expression, and the insect cell hosts have some eucaryotic protein processing capabilities. Also, while insect cells remain more expensive to cultivate than bacteria, recent developments have significantly reduced the cost of producing foreign gene products in this system. Based on these properties, the baculovirus-insect cell system is a widely used tool for the production of foreign gene products, particularly eucaryotic proteins that must be co- and post-translationally processed (Summers and Smith, 1987, Luckow and Summers, 1988; Miller, 1988; O'Reilly et al., 1992).
However, a major limitation of using the baculovirus-insect cell system for recombinant glycoprotein production is that the N-glycosylation pathway in insect cells differs from the pathway found in higher eukaryotes (Jarvis and Summers, 1992; Kornfeld and Kornfeld, 1985). This is a significant drawback as there is increasing evidence that proper glycosylation imparts important functions to many eukaryotic proteins (Welply, 1991).
Most of the information on the N-glycosylation pathway in insect cells has come from structural studies on foreign glycoproteins expressed in baculovirus-infected cell lines or in larvae (reviewed by Jarvis and Summers, 1992; O'Reilly et al., 1992; Jarvis, 1993a). These studies have demonstrated that insect cells have processing glucosidases and mannosidases which convert high mannose oligosaccharides to trimmed structures with as few as three mannose residues. Several lines of evidence indicate that these cells also have a fucosyltransferase that can add fucose to the core Asn-linked GlcNAc residue (Staudacher et al., 1992).
However, mammalian cells extend such trimmed oligosaccharide structures by adding N-acetylglucosamine, galactose, fucose, and sialic acid residues to produce a complex biantennary structure containing penultimate galactose and terminal sialic acid residues (Kornfeld and Kornfeld, 1985; Paulson and Colley, 1989; Moremen et al., 1994). Insect cells generally do not produce these extended complex structures, indicating that the requisite processing activities are either absent or too low to be generally effective in these cells. This limits the current usefulness of insect cells. Although some recent studies indicate that insect cell lines can produce glycoproteins with certain terminal glycosylation patterns more similar to those found in higher eukaryotes (Kubelka et al., 1994; Ackermann et al., 1995, Ogonah, et al., 1996, Davidson et al., 1990; Davidson and Castellino, 1991a), the vast majority of recombinant proteins produced in insect cells lack these structures.
SUMMARY OF THE INVENTION
The present invention overcomes the drawbacks in the prior art by providing new and improved baculoviral expression vectors, insect cell lines, compositions and various methods of use. The invention first provides a baculovirus expression vector characterized as either: (a) comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene operatively positioned under the control of and in frame with a promoter; or (b) comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene operatively positioned under the control of a baculoviral immediate early, delayed early, early or late promoter.
Recombinant vectors for important aspects of the present invention. The term “expression vector or construct” means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of a RNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense or ribozyme constructs. It will naturally be understood that the transcriptional units each comprise the appropriate transcription and translation initiation and termination signals, such as ATG start signals, and are positioned in the proper orientation to allow transcription of the gene.
Where the present invention comprises the vector of part (a) above, it will be understood that the vector may a vantageously further include a third, fourth, fifth, sixth, seventh, eighth and/or a ninth glycosylation enzyme transcriptional unit, the third, fourth, fifth, sixth, seventh, eighth and/or a ninth transcriptional unit comprising a structural gene for a third, fourth, fifth, sixth, seventh, eighth and/or a ninth oligosaccharide processing enzyme, operatively positioned under the control of a promoter.
Equally, where the present invention comprises the vector of part (b) above, the vector may also further include a second, third, fourth, fifth, sixth, seventh, eighth and/or a ninth glycosylation enzyme transcriptional unit, the second, third, fourth, fifth, sixth, seventh, eighth and/or a ninth transcriptional unit comprising a structural gene for a second, third, fourth, fifth, sixth, seventh, eighth and/or a ninth oligosaccharide processing enzyme, operatively positioned under the control of a promoter.
Exemplary oligosaccharide processing enzymes for use in the invention include, but are not limited to &agr;-glucosidases, including &agr;-glucosidase I and &agr;-glucosidase I, &agr;-mannosidases, such as &agr;-mannosidase I and &agr;-mannosidase II, N-acetylglucosaminyltransferases, including, but not limited to N-acetylglucosaminyltransferase and N-acetylglucosaminyltransferase II, fucosyltransferases, galactosyltransferases and sialyltransferases. The oligosaccharide processing enzymes contemplated for use in the present invention include, but are not limited to, the extensive list provided herein below in Table 1.
The oligosaccharide processing enzymes may be used individually, or in any combination. In certain preferred embodiments, the oligosaccharide processing enzyme will be a
Crouch Deborah
Fulbright & Jaworski L.L.P.
University of Wyoming
Woitach Joseph T.
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