Expression Vector

Abstract

An expression vector including two separately inducible converging promoters P1 and P2, and expression system including such an expression vector and an additional regulator vector, a method of protein expression using such an expression system, and a method of investigating (meta)genome libraries using such an expression system.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of international patent application no. PCT / EP2009 / 008977, filed Dec. 15, 2009, designating the United States of America, and published in German on Jul. 8, 2010 as WO 2010 / 075956, the entire disclosure of which is incorporated herein by reference. Priority is claimed based on European Patent Application no. EP 08021794.6, filed Dec. 16, 2008, which likewise is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]The invention relates to an expression vector that is suitable for efficient screening of (meta)genome libraries, preferably in Escherichia coli. [0003]Only about 1-5% of all known microorganisms are at present cultivable in the laboratory with current methods. Methods have been developed in recent times which should make it possible to use the genetic resources of non-cultivable microorganisms. This field is also called “metagenomics”, with the term “metagenome” denoting the geneti...

AI Technical Summary

Benefits of technology

[0019]Another object is to provide an expression system that is characterized by a high cloning efficiency linked to efficient, controllable expression.

[0024]It was found, surprisingly, that the expression vector according to the invention is particularly suitable for activity screening of (meta)genome libraries, as both directions of reading are covered. The loss of half of the sequence information contained in the (meta)genome library or the need to screen double the number of clones, as must be accepted when using conventional expression vectors, can be avoided by the expression vector according to the invention.

Problems solved by technology

Problems with the metagenome technique relate in particular to expression of the genes found.

These include inadequate transcription, for example because promoters are not recognized, toxicity of the products to the host, missing cofactors or chaperones and therefore incorrect folding of the proteins in the heterologous host, and missing secretion systems (W. R. Streit et al., Curr Opin Microbiol.

Owing to the weakness of the promoter or the non-recognition of non-E. coli promoters, some of the target proteins are barely expressed, or not at all, so that activity screening of the target proteins is far more difficult.

These limitations make iterative activity screening of sub-libraries (cluster screening, cf.

Instead, complicated and time-consuming activity screening with individual clones, e.g. on agar plates, is necessary.

Another problem in activity screening is that when constructing (meta)genome libraries it is not possible to influence the orientation of the open reading frame (ORF) on the cloned DNA.

In activity screening with conventional expression vectors, a large part of the sequence information contained in the (meta)genome library is therefore often lost because the promoter used only covers one of the two possible directions of reading.

In this system, however, the DNA to be cloned into the insertion sequence is not under the control of both promoters.

Such a system is hardly suitable for screening metagenome libraries, as only 50% of the sequence information contained is captured.

However, that method does not use a vector with promoters that are inducible separately from one another, and flow towards one another.

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