Compositions and Methods for Controlling Copy Number for a Broad Range of Plasmids and Uses Thereof

Abstract

The present invention provides compositions and methods for controlling the copy number for a broad range of plasmids and uses thereof. Disclosed is a host cell for conditional control of copy number of a plasmid, which host cell comprises a poly(A) polymerase gene that is operably joined to a conditionally inducible promoter, and a method for cloning and stably maintaining a DNA sequence encoding a heterologous polypeptide in the host cell.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a continuation application of U.S. patent application Ser. No. 10 / 883,459, filed Jul. 1, 2004, which claims priority to U.S. Provisional Patent Application Ser. No. 60 / 483,955, filed Jul. 1, 2003. The entire disclosure of all priority applications is specifically incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]Not Applicable.FIELD OF THE INVENTION[0003]This invention relates to production and use of host cells for control of replication of plasmids, including, but not limited to, DNA vectors, in the host cells, and to their use for production of DNA, RNA and / or polypeptides encoded by DNA contained in these plasmids.BACKGROUND OF THE INVENTION[0004]Plasmids are normally circular, extrachromosomal DNA molecules that replicate autonomously within the cells of host organisms. The cells of many unicellular organisms, including some bacteria, contain naturally occurring w...

AI Technical Summary

Benefits of technology

The present invention provides host cells and methods for controllably changing the copy number of a plasmid that is introduced into a host cell. This is achieved by inducing the poly(A) polymerase gene, which is operably joined to a conditional inducible promoter. The invention allows for low-copy replication of plasmid vectors with ori's of the present invention, and also enables high-copy replication under different conditions. The invention also allows for the production of toxic or detrimental polypeptides in a host cell, and can be used for cloning and stable maintenance of difficult-to-clone sequences. The host cells can produce large quantities of heterologous polypeptides, even toxic ones.

Problems solved by technology

This can lead to undesirable results, however, since many such high copy number plasmids tend not to be maintained stably within transformed cells and may be lost from the cells before they can be grown to sufficient levels to permit bulk production of foreign products.

For example, the foreign product may be toxic and / or inhibit propagation of the transformed cells, or the high copy number plasmids themselves may be inherently unstable or recombine with DNA sequences in other copies of the recombinant plasmid that are present in the same cell.

The removal of inhibitor requires complicated manipulations and is not always possible.

However, temperature-dependent copy number may be limited to particular mutant plasmids, which may or may not contain suitable restriction enzyme cloning sites for a particular foreign DNA sequence.

Also, introduction of copy number temperature dependence may introduce a source of instability into the plasmid, and these mutant plasmids may be unstable or subject to loss when cells carrying them are propagated over a prolonged period of time.

Another disadvantage of this approach is the fact that higher temperatures may have a negative impact on protein stability.

While the compositions and methods disclosed in the art provide solutions for controlling copy number of recombinant plasmids for particular applications, they suffer from certain disadvantages.

All of the methods are limited to vectors having particular additional genetic elements, genes or other modifications.

The methods of Yarranton et al. and of Szybalski et al. require the use of vectors with two origins of replication, which increases the size of the vector and in most cases will limit the number and kind of restriction enzyme sites available for cloning of foreign genes.

The method of Sagawa is limited to vectors that contain particular inducible RNAII-encoding DNA sequences for high-copy replication of the vector and, in most cases, an inducible RNA polymerase gene for transcription of a foreign gene that is cloned in the vector.

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