Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1998-10-15
2001-03-27
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S006120, C435S069100
Reexamination Certificate
active
06207442
ABSTRACT:
BACKGROUND OF THE INVENTION
Recombinant DNA technology is now widely used for both research and commercial protein production. An essential tool of this technology is the plasmid, a double-stranded DNA molecule that can replicate (autonomously or by chromosomal integration) in one or more species of host cell. A DNA sequence of interest can be inserted into a plasmid and replicated in large quantities. If a sequence encoding a protein is operably linked to a transcription promoter, the sequence can be expressed and the encoded protein can be recovered from the cell.
DNA sequences of interest are commonly joined to plasmids by cutting both pieces of DNA with a restriction endonuclease to provide complementary ends that are then enzymatically ligated together. If the sequences of the two pieces do not permit the use of a single restriction endonuclease, small oligonucleotide adapters can be used to join the free ends.
Advances in recombinant DNA technology, including the use of automation, have resulted in the rapid identification and production of novel DNA sequences. To further characterize these sequences it is necessary to express them and study the properties of the encoded proteins. Expression generally requires the precise joining of DNA sequences within expression vectors to maintain the functional relationships between genetic elements (e.g., open reading frame, promoter function, etc.). Current methods, which rely primarily on the use of restriction enzymes, can be problematic when it is necessary to rapidly express a large number of sequences and analyze their products because these methods can require extensive and time-consuming manipulation of sequences to obtain the desired junctions. There is thus a need in the art for improved methods of plasmid construction. Toward this end, the present invention provides a standardized plasmid into which a variety of DNA sequences can be readily inserted and subsequently expressed, as well as related cloning methods and other improvements.
SUMMARY OF THE INVENTION
The invention provides a universal acceptor plasmid that can be used to clone a DNA encoding any polypeptide of interest, including polypeptide fusions. The acceptor plasmid is useful within methods for preparing double stranded, circular DNA molecules. One such method comprises the steps of: (a) providing a double-stranded donor DNA fragment encoding a polypeptide of interest; (b) providing a double-stranded, linear acceptor plasmid having blunt first and second ends and comprising a selectable marker and replication sequence that are functional in
Saccharomyces cerevisiae,
wherein the acceptor plasmid is essentially free of DNA encoding the polypeptide of interest; (c) providing a first double-stranded DNA linker comprising a first segment identical in sequence to a first region of the acceptor plasmid and a second segment identical in sequence to a first region of the donor DNA fragment, wherein each of the first and second segments of the first linker is at least 10 bp in length, preferably at least 50 bp in length; (d) providing a second double-stranded DNA linker comprising a first segment identical in sequence to a second region of the acceptor plasmid and a second segment identical in sequence to a second region of the donor DNA fragment, wherein each of the first and second segments of the second linker is at least 10 bp in length, preferably at least 50 bp in length; and (e) combining the donor DNA fragment, acceptor plasmid, first DNA linker, and second DNA linker in a
Saccharamyces cerevisiae
host cell whereby the donor DNA fragment is joined to the acceptor plasmid by homologous recombination of the donor DNA, acceptor plasmid, and linkers to form a closed, circular plasmid comprising a region encoding the polypeptide of interest. Within one embodiment of the invention, the acceptor plasmid further comprises a transcription promoter proximal to the first end, and the donor DNA fragment is operably linked to the transcription promoter within the closed, circular plasmid. Within a related embodiment, the acceptor plasmid further comprises a transcription terminator proximal to the second end, and the donor DNA fragment is operably linked to the transcription terminator within the closed, circular plasmid. Within other embodiments, the acceptor plasmid further comprises a DNA segment encoding a leader peptide and/or one or more DNA segments encoding a peptide tag, positioned such that these DNA segments are operably linked to the donor DNA fragment within the closed, circular plasmid. Within a preferred embodiment, the acceptor plasmid further comprises (a) a promoter, a DNA segment encoding a leader peptide, and a DNA segment encoding a first peptide tag, wherein the DNA segment encoding a leader peptide is positioned between the promoter and the DNA segment encoding a first peptide tag proximal to the first end of the acceptor plasmid, and wherein the promoter, DNA segment encoding a leader peptide, and DNA segment encoding a first peptide tag are operably linked; and (b) a DNA segment encoding a second peptide tag proximal to the second end of the acceptor plasmid.
A related aspect of the invention provides a method for preparing a double stranded, circular DNA molecule comprising the steps of: (a) providing a plurality of overlapping, double-stranded donor DNA fragments which collectively encode a polypeptide of interest; (b) providing a double-stranded, linear acceptor plasmid having blunt first and second ends and comprising a selectable marker and replication sequence that are functional in
Saccharamyces cerevisiae
, wherein the acceptor plasmid is essentially free of DNA encoding the polypeptide of interest; (c) providing a first double-stranded DNA linker comprising a first segment identical in sequence to a first region of the acceptor plasmid and a second segment identical in sequence to a region of one of the donor DNA fragments, wherein each of the first and second segments of the first linker is at least 10 bp in length, preferably at least 50 bp in length; (d) providing a second double-stranded DNA linker comprising a first segment identical in sequence to a second region of the acceptor plasmid and a second segment identical in sequence to a region of another of the donor DNA fragments, wherein each of the first and second segments of the second linker is at least 10 bp in length, preferably at least 50 bp in length; and (e) combining the donor DNA fragments, acceptor plasmid, first DNA linker, and second DNA linker in a
Saccharamyces cerevisiae
host cell whereby the donor DNA fragments are joined to the acceptor plasmid by homologous recombination of the donor DNA fragments, acceptor plasmid and linkers to form a closed, circular plasmid comprising a region encoding the polypeptide of interest. Within certain embodiments of the invention, the acceptor plasmid further comprises one or more of a transcription promoter, a transcription terminator, a DNA segment encoding a leader peptide, and one or more DNA segments encoding a peptide tag, as disclosed above.
These and other aspects of the invention will become evident upon reference to the following detailed description and figures.
REFERENCES:
patent: 5721367 (1998-02-01), Kay et al.
patent: 5989866 (1999-11-01), Deisher et al.
patent: 0288435A1 (1988-10-01), None
patent: 97/07206 (1997-02-01), None
Ma et al., “Plasmid construction by homologous recombination in yeast,” 1987, Gene, 58, pp. 201-216.*
Hopp et al., “A Short Polypetide Marker Sequence Useful for Recombinant Protein Identification and Purification,” 1988, Bio/Technology, vol. 6, pp. 1204-1210.*
Ma et al.,Gene58: 201-216, 1987.
Oldenburg et al.,Nucleic Acids Res.25: 451-452, 1997.
Patrusky,Mosaic21: 44-52, 1990.
Pompon et al.,Gene83: 15-24, 1989.
Hudson, Jr. et al.,Genome Res. 7: 1169-1173, 1997.
Brusca John S.
Kim Young
Parker Gary E.
ZymoGenetics Inc.
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