Signal sequence trapping

DETAILED DESCRIPTION OF THE INVENTION The present invention allows the screening of previously established genebanks or libraries by proxy, for genes encoding secreted polypeptides or enzymes even of unknown activity and thus without known screening assays.
The method of the invention enables screening for polypeptides of potential industrial interest that would not likely have been isolated using conventional screening assays.
A method for identifying and isolating a gene of interest from a gene library, wherein the gene encodes a polypeptide carrying a signal sequence for secretion or partial secretion, the method comprising the steps of: (a) providing a genomic DNA library or a cDNA library; (b) inserting into said library a DNA fragment comprising a promoterless and secretion signal-less polynucleotide encoding a secretion reporter; (c) introducing the library comprising the inserted DNA fragment into a host cell; (d) screening for and selecting a host cell that secretes or partially secretes the active secretion reporter; (e) identifying the gene of interest into which the secretion reporter was inserted in the selected host cell, by sequencing the DNA flanking the inserted DNA fragment; and (f) isolating the complete gene of interest identified in step e).
The present invention can be performed using any gene libraries known in the art, specifically it can also be used with gene libraries of viable but non-culturable organisms as typically seen in environmental samples.
Processes of producing representative or normalized gene-libraries from environmental samples containing non-culturable organisms have been described in the art (U.
S.
Pat.
No.
5,763,239).
Accordingly a preferred embodiment of the present invention relates to a method of the first aspect, wherein the complete gene of interest in step (f) is isolated from the library of step (a).
In the art several ways of inserting a DNA fragment into a genome are known, one example is insertion by transposition, however this usually requires time-and labour consuming mating experiments to be carried out