Abstract: The present invention provides methods for identifying a factor which modulates gene expression comprising providing a library of at least 1×106 nucleic acid molecules each comprising a stochastic sequence of at least 50 nucleotides, introducing said nucleic acid molecules into nucleic acid constructs which comprise a reporter sequence and which do not comprise a further separate sequence upstream of the reporter sequence that can modulate expression of the reporter sequence to generate test constructs, introducing test constructs into host cells and assessing expression of the reporter sequence, thereby to identify a factor which modulates gene expression. Libraries of nucleic acid molecules, test constructs and host cells for use in such methods are also provided.

Abstract: The present invention provides a synthetic mRNA leader for enhancing the expression of a gene encoding a desired protein, vectors comprising said synthetic mRNA leader and methods of producing a desired gene product using said synthetic mRNA leader and vector, said method comprising expressing said gene using a synthetic mRNA leader which comprises from 5? to 3?: (i) a first mRNA leader sequence element; (ii) a spacer region; and (iii) a second mRNA leader sequence element; wherein said first mRNA leader sequence element is a modified transcription-stimulating mRNA leader capable of enhancing transcription of a gene relative to an unmodified reference mRNA leader sequence and/or said second mRNA leader element is a modified translation-stimulating mRNA leader capable of enhancing the translation of a gene transcript relative to an unmodified reference mRNA leader sequence.

Abstract: The present invention concerns a method of producing a desired gene product in a recombinant gene expression system, said method comprising expressing said gene from a Pm promoter-based expression system using at least two mutant elements selected from: (i) a mutant Pm promoter; (ii) a mutant mRNA leader; and (iii) a mutant XyIS; wherein said mutant elements each comprise one or more mutations which enhance expression of said desired gene. Particularly combinations of a mutant Pm promoter and a mutant mRNA leader are concerned. Isolated nucleic acid molecules, vectors, host cells, libraries, expression systems, methods of enhancing expression, obtaining nucleic acid molecules and identifying combination mutants which enhance expression, artificially constructed operons and their uses are also encompassed.

Abstract: The present invention provides a synthetic mRNA leader for enhancing the expression of a gene encoding a desired protein, vectors comprising said synthetic mRNA leader and methods of producing a desired gene product using said synthetic mRNA leader and vector, said method comprising expressing said gene using a synthetic mRNA leader which comprises from 5? to 3?: (i) a first mRNA leader sequence element; (ii) a spacer region; and (iii) a second mRNA leader sequence element; wherein said first mRNA leader sequence element is a modified transcription-stimulating mRNA leader capable of enhancing transcription of a gene relative to an unmodified reference mRNA leader sequence and/or said second mRNA leader element is a modified translation-stimulating mRNA leader capable of enhancing the translation of a gene transcript relative to an unmodified reference mRNA leader sequence.

Abstract: The present invention concerns a method of producing a desired heterologous gene product wherein said heterologous gene product is expressed from a strong promoter, said method comprising expressing said gene using a mutant mRNA leader which comprises one or more mutations which enhance transcription of the gene. The invention also provides a mutant Pm mRNA leader sequence, and a vector and a library comprising the leader sequence. Methods of obtaining an mRNA mutant leader and identifying an mRNA mutant leader are encompassed, along with a vector for selection or identification of an mRNA leader mutant and a use thereof for screening.

Abstract: The present invention concerns a method of producing a desired heterologous gene product wherein said heterologous gene product is expressed from a strong promoter, said method comprising expressing said gene using a mutant mRNA leader which comprises one or more mutations which enhance transcription of the gene. The invention also provides a mutant Pm mRNA leader sequence, and a vector and a library comprising the leader sequence. Methods of obtaining an mRNA mutant leader and identifying an mRNA mutant leader are encompassed, along with a vector for selection or identification of an mRNA leader mutant and a use thereof for screening.

Abstract: The present invention concerns a method of producing a desired gene product in a recombinant gene expression system, said method comprising expressing said gene from a Pm promoter-based expression system using at least two mutant elements selected from: (i) a mutant Pm promoter; (ii) a mutant mRNA leader; and (iii) a mutant XyIS; wherein said mutant elements each comprise one or more mutations which enhance expression of said desired gene. Particularly combinations of a mutant Pm promoter and a mutant mRNA leader are concerned. Isolated nucleic acid molecules, vectors, host cells, libraries, expression systems, methods of enhancing expression, obtaining nucleic acid molecules and identifying combination mutants which enhance expression, artificially constructed operons and their uses are also encompassed.