Bidirectional promoter
The invention refers to a library of bidirectional expression cassettes or expression vectors comprising a repertoire of bidirectional promoter sequences, each expression cassette comprising a promoter sequence operably linked to a first gene in one direction, and operably linked to an oppositely oriented second gene in the other direction which is different from the first gene, and bidirectional Pichia pastoris or CHO cells promoter sequences. The invention further refers to a method of screening or selecting a bidirectional promoter suitable for expressing at least two GOI in a host cell and a kit comprising a) an expression cassette consisting of the first and second genes and a stuffer sequence separating them, which stuffer sequence comprises a recognition site for a type IIS restriction enzyme at both ends; b) the type IIS restriction enzyme; c) and a repertoire of promoter, preferably a promoter library including bidirectional promoters.
1. A library of bidirectional expression cassettes comprising a plurality of bidirectional promoter sequences, each expression cassette comprising a bidirectional promoter sequence operably linked to a first gene in one direction, and operably linked to an oppositely oriented second gene in the other direction which is different from the first gene, wherein the bidirectional promoter sequences are functional in a Pichia pastoris or CHO cell, and wherein the bidirectional promoter sequences comprise two sequences selected from the group consisting of (i) SEQ ID NO:39-78, (ii) SEQ ID NO: 126-135, and (iii) SEP ID NO: 136-165.
2. A library of expression vectors each comprising at least one expression cassette as defined in claim 1 .
3. The library of claim 1 , wherein the genes comprise a gene of interest (GOI) or a reporter gene, and wherein (i) the genes encode protein components of a composite protein or protein complex, (ii) the first gene encodes a first protein which supports folding or targeting of a second protein, (iii) the genes are of the same metabolic or regulatory pathway, or (iv) the genes are of different pathways wherein one pathway supports other pathways.
4. The library of claim 1 , wherein the plurality of bidirectional promoter sequences comprises at least 50 different bidirectional promoter sequences.
5. A method of screening or selecting a bidirectional promoter suitable for expressing at least two GOI in a host cell which comprises
a) providing the library of claim 1 , comprising the at least two GOI as the first and second genes;
b) selecting a library member which has a proven bidirectional transcription activity; and
c) identifying the bidirectional promoter sequence comprised in the selected library member and/or using the same for producing an expression construct to express said at least two GOI under the transcriptional control of said bidirectional promoter sequence.
6. The method according to claim 5 , wherein the transcription activity is qualitatively and/or quantitatively determined.
7. The method of claim 5 , wherein the library member is selected according to the transcription activity of the first and second genes, which is differently regulated, preferably any of a constitutive activity, or activity induced or de-repressed by a carbon source.
8. The library of claim 3 , wherein the composite protein is a heterodimeric protein.
9. The library of claim 3 , wherein the protein complex is formed by interaction of the protein components.