Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-04-23
2002-10-29
McKelvey, Terry (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...
C435S243000, C435S320100
Reexamination Certificate
active
06472177
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
This invention relates to processes and expression vectors for producing and recovering heterologous polypeptides from bacterial cells. More particularly, the invention relates to expression vectors that comprise polynucleotide control sequences that permit simultaneous or independent control over both the level at which the polypeptide-encoding polynucleotide sequences are transcribed and the copy number of the expression vectors.
In a typical expression system, heterologous polypeptide production is either not regulated or is regulated by modulating transcription from a transcriptional promoter operably linked upstream of a polynucleotide that encodes the heterologous polypeptide. To ensure that large amounts of protein are produced, expression vectors having high-copy-number are typically employed. In such high-copy-number expression systems, when the promoter that regulates transcription of the polypeptide-encoding sequence is not tightly regulated (is “leaky”), even small amounts of the polypeptide produced can be toxic to, or can have other adverse effects upon, the host cell. Also, high-copy-number expression vectors can be unstable and can yield undesired deletions or mutations or chimeric recombination products. This is a rather common disadvantage of high-copy-number vectors.
Alternatively, a host cell can better tolerate transcriptional leakage when the expression vector is present in one or a few copies per cell. However, such vectors are also of limited utility in that the amount of polypeptide produced from a single-copy vector is very small and large numbers of cells containing such a vector carrying the polynucleotide of interest must be grown to obtain sufficient quantities of the polypeptide for isolation and purification.
Bacterial artificial chromosome (BAC) vectors (and plasmid forms, pBAC) are single-copy vectors used to maintain large genomic DNA fragments, and have not been used as expression vectors. BAC (or pBAC) vectors typically accommodate inserts in the range of up to 300 kilobase pairs. Kim, U-J et al., “Construction and Characterization of a Human Bacterial Artificial Chromosome Library,” Genomics 34:213-218 (1996) describe a now widely used BAC cloning vector, pBeloBAC11, that uses lacZ X-Gal/IPTG complementation to distinguish by color insert-containing recombinant molecules from colonies carrying the BAC vector without an insert. The pBeloBAC11, is an improvement over pBAC 108L, a prior BAC cloning vector described in Shizuya, H., “Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in
Escherichia coli
using an F-factor-based vector,” P.N.A.S. U.S.A. 89:8794-8797 (1992) that lacked the ability to identify insert-containing BACs. U.S. Pat. No. 5,874,259 (incorporated herein as if fully set forth in its entirety) discloses conditionally amplifiable BAC vectors having, in addition to an F factor-based origin of replication that maintains the vector at one copy per cell, a conditional origin of replication at which replication is initiated in response to a suitable signal in the host cell. The disclosed vectors facilitate obtaining large amounts of a genomic polynucleotide fragment, thereby overcoming a known disadvantage of BAC vectors, namely low DNA yield. After induction, the copy number of the vector or the insert-containing vector increases substantially and the polynucleotides of interest can be isolated. In the vectors of U.S. Pat. No. 5,874,259, a pair of excision-mediating sites (EMS) can optionally flank the conditional ori and a site into which a cloned genomic polynucleotide fragment can be cloned. In the latter case, the nucleic acid between the EMS can be excised to create a circular plasmid that comprises the genomic fragment insert and which can replicate when the conditional ori is activated by induced Rep protein. None of the aforementioned patent and publications contemplate employing BAC vectors for transcribing polynucleotides and none of the vectors includes all of the elements for doing so. While pBeloBAC11 contains the T7 and SP6 promoters, these are used only as sites to anneal the sequencing primers, not as promoters of transcription.
BRIEF SUMMARY OF THE INVENTION
In a first aspect, the present invention is an expression vector for expressing in compatible host cells a heterologous polypeptide encoded by a heterologous polynucleotide sequence in the vector. The expression vector of the invention comprises both a conditionally amplifiable origin of replication (ori) that functions in the host cells and a tightly regulated inducible transcriptional promoter operably linked to the heterologous polynucleotide sequence. The vector is capable of independent replication in the host cell and therefore also can include polynucleotide sequences that encode any proteins required for plasmid replication, maintenance, and partitioning that are not otherwise provided in the host cells, either in the growth medium, in the host genome or on a separate expression vector.
For convenience, the expression vector also preferably contains a selectable marker for confirming the presence in the host cells of the vector. The vector also preferably includes at least one cloning site into which the heterologous polynucleotide of interest can be cloned. It is also desirable to distinguish clones that contain a heterologous polynucleotide of interest from the expression vector itself. In accord with conventional practice, the cloning site can therefore be situated in a larger polynucleotide coding sequence that confers an alterable phenotype upon the host cells. A change in that phenotype can indicate that a heterologous polynucleotide is present in the cloning site. An expression vector of the invention can optionally include a pair of excision mediation sites (EMS) flanking the conditional ori, the inducible promoter and the site into which the polynucleotide of interest is inserted, for excision of the expression cassette from the vector to a separate, smaller, and conditionally-amplifiable plasmid.
In a related aspect, the invention is also summarized in that a host cell for producing heterologous polypeptides includes in its interior an expression vector of the present invention. The host cell can conditionally provide the signals required to activate the conditional ori and/or the inducible promoter in the modified vector. The expression vector in the host cell can comprise a heterologous polynucleotide insert that encodes the polypeptide of interest.
In yet another related aspect, the invention is further summarized in that a method for obtaining a desired amount of the heterologous polypeptide of interest from a host cell that contains the expression vector with heterologous polynucleotide insert includes the steps of modulating the activity in the host cell of an agent that conditionally amplifies the ori to maintain the vector at a desired copy number and modulating the activity of an agent that induces a desired transcription level of the heterologous polynucleotide to maintain an overall desired level of polypeptide production from the vector, and isolating the heterologous polypeptide from the host cells.
It is an object of the present invention to produce heterologous polypeptides in a host cell, even where the polypeptide is toxic or has other adverse effects on the host cell that would prevent cloning and/or stable maintenance of inserted polynucleotide prior to the overproduction of the polypeptide in conventional cell-based protein expression systems.
It is another object of the present invention to retain the advantageous properties of existing vectors including stability of an inserted polynucleotide.
It is a feature of the present invention that the vector includes both a conditional origin of replication and an inducible transcriptional promoter.
It is a feature of the present invention that the expression vector allows user control over b
Hradecna Zdenka
Szybalski Waclaw T.
Wild Jadwiga
McKelvey Terry
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
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