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WARF: P01037US

Expression Vector with Dual Control of Replication and Transcription


INVENTORS -

Waclaw Szybalski, Jadwiga Wild, Zdenka Hradecna

The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing a controlled BAC expression system that can produce large amounts of genomic polypeptide upon induction.
OVERVIEWHigh copy number vectors for producing large quantities of recombinant proteins can often be unstable or toxic to the host if gene expression is not tightly regulated. Bacterial artificial chromosomes (BACs) are widely used in genomics because these single-copy vectors are more stable than multi-copy vectors and can maintain large inserts. However, as single-copy expression systems, their yield of DNA is very low.
THE INVENTIONUW-Madison researchers have constructed a controlled BAC expression system that can produce large amounts of genomic polypeptide upon induction. Their modified pBAC system includes both a conditionally amplifiable origin of replication(oriV) with a broad host range, and a tightly regulated, inducible transcriptional promoter linked to a gene of interest. The conditional oriV and the inducible promoter can be activated jointly or separately by signals in the host cell.

Specifically, BAC copy number is controlled by a single inducing protein, TrfA, which induces replication from oriV. Gene transcription is controlled by an arabinose-inducible promoter, which can be induced to produce many copies of the inserted gene. Thus, this pBAC includes all of the elements necessary for transcribing large quantities of polynucleotides in a controlled fashion.
APPLICATIONS
  • Production of large quantities of recombinant proteins
KEY BENEFITS
  • This modified BAC provides dual control of both transcriptional level and copy number, allowing for tight modulation of gene expression.
  • The re-engineered BAC allows conditional, “on command only” amplification of the vector, including the cloned DNA.
  • Produces very low background and “leakage” of potentially toxic cloned gene products
  • Upon induction with arabinose, the copy number of the inserted gene can increase from one up to 100.
  • Can accommodate large nucleic acid inserts
Contact Information
For current licensing status, please contact Jennifer Gottwald at jennifer@warf.org or 608-960-9854.
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