Uncoupling of DNA insert propagation and expression of protein for phage display
The present invention provides an advance in phage display technology by permitting the uncoupling of the propagation of phages containing inserted sequences encoding heterologous polypeptides from the expression of said polypeptides. The invention provides phage constructs and methods for their use to permit phage coat protein expression, and thus phage propagation, in the absence of display of heterologous polypeptides, which may be expressed as a fusion with said coat protein in a regulated manner.
1. An expression system comprising
a modified T7 phage genome comprising a first nucleic acid molecule comprising a promoter and/or regulatory region operably linked, in a 5′ to 3′ orientation, to a sequence encoding a T7 phage coat protein operably linked in a 5′ to 3′ orientation to a sequence comprising a termination codon operably linked in a 5′ to 3′ orientation to a sequence encoding a heterologous polypeptide and
a suppressor construct comprising an inducible promoter and/or regulatory region operably linked to a sequence encoding a suppressor tRNA corresponding to said termination codon.
2. The system of claim 1 wherein said promoter and/or regulatory region is endogenous to a T7 phage genome.
3. The system of claim 1 wherein said promoter and/or regulatory region is an endogenous T7 promoter.
4. The system of claim 1 wherein said inducible promoter and/or regulatory region is the arabinose BAD promoter and the araC regulatory gene.
5. The system of claim 1 wherein said sequence comprising said termination codon further comprises a linker sequence encoding an enzymatic cleavage site.
6. The system of claim 1 wherein said suppressor construct further comprises an expressible sequence encoding chloramphenicol resistance.
7. The system of claim 1 wherein said termination codon is selected from UAA and UGA.
8. The system of claim 1 wherein said termination codon is UAG.
9. The system of claim 1 wherein said suppressor tRNA is tRNA Ala or tRNA Glu .
10. The system of claim 1 wherein said suppressor tRNA is tRNA Ala .
11. The system of claim 5 wherein said enzymatic cleavage site is recognized and cleaved by an enzyme selected from the group consisting of subtilisin, H64A subtilisin, Generase I, tobacco etch virus (TEV) protease, thrombin, factor Xa, and enterokinase.