Uncoupling of DNA insert propagation and expression of protein for phage display
View Patent ↗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. A method of making a heterologous polypeptide comprising:
(i) propagating a modified T7 phage in an E. coli cell,
wherein the modified T7 phage comprises, in a 5′ to 3′ orientation, a promoter nucleic acid sequence, which is operably linked to a nucleic acid sequence encoding a surface protein of the modified T7 phage, which is operably linked to a nucleic acid sequence comprising a termination codon, which is operably linked to a nucleic acid sequence encoding the heterologous polypeptide;
wherein the E. coli cell comprises a suppressor construct comprising an inducible promoter nucleic acid sequence, which is operably linked in a 5′ to 3′ orientation to a nucleic acid sequence encoding a suppressor tRNA that binds to the termination codon of the modified T7 phage;
wherein expression of the suppressor tRNA is not induced; and
(ii) inducing the inducible promoter of the suppressor construct; whereby the suppressor tRNA is expressed in the E. coli cell so that the heterologous polypeptide is expressed and displayed on the surface of the modified T7 phage.
2. A method of making a heterologous polypeptide comprising:
(i) propagating a modified T7 phage in a first E. coli cell,
wherein the modified T7 phage comprises, in a 5′ to 3′ orientation, a promoter nucleic acid sequence, which is operably linked to a nucleic acid sequence encoding a surface protein of the modified T7 phage, which is operably linked to a nucleic acid sequence comprising a termination codon, which is operably linked to a nucleic acid sequence encoding the heterologous polypeptide;
wherein the first E. coli cell does not contain a suppressor tRNA that can bind to the termination codon of the modified T7 phage;
(ii) introducing the modified T7 phage of step (i) into a second E. coli cell, which comprises a suppressor construct comprising an inducible promoter nucleic acid sequence, which is operably linked in a 5′ to 3′ orientation to a nucleic acid sequence encoding a suppressor tRNA that binds to the termination codon of the modified T7 phage; and
(iii) inducing the inducible promoter of the suppressor construct; whereby the suppressor tRNA is expressed in the second E. coli cell so that the heterologous polypeptide is expressed and displayed on the surface of the modified T7 phage.
3. A method of making a heterologous polypeptide comprising:
(i) propagating a modified T7 phage in a first E. coli cell, wherein the modified T7 phage comprises, in a 5′ to 3′ orientation, a promoter nucleic acid sequence, which is operably linked to a nucleic acid sequence encoding a surface protein of the phage, which is operably linked to a nucleic acid sequence comprising a termination codon, which is operably linked to a nucleic acid sequence encoding the heterologous polypeptide;
wherein the first E. Coli cell does not contain a suppressor tRNA that can bind to the termination codon of the phage;
(ii) introducing the modified T7 phage of step (i) into a second E. coli cell, which comprises a suppressor construct comprising a modified T7 phage promoter nucleic acid sequence, which is operably linked in a 5′ to 3′ orientation to a nucleic acid sequence encoding a suppressor tRNA that binds to the termination codon; so that the suppressor tRNA is expressed in the second E. coli cell and the heterologous polypeptide is expressed and displayed on the surface of the modified T7 phage.
4. The method of claim 1 or 2 , wherein the promoter of the phage is a T7 modified T7 phage promoter.
5. The method of claim 1 , 2 or 3 , wherein the modified T7 phage further comprises a regulatory region nucleic acid sequence operably linked upstream of the nucleic acid sequence encoding the surface protein of the modified T7 phage.
6. The method of claim 5 , wherein the suppressor construct further comprises a regulatory region nucleic acid sequence operably linked upstream of the nucleic acid sequence encoding the suppressor tRNA.
7. The method of claim 1 , 2 or 3 , wherein the suppressor construct further comprises a regulatory region nucleic acid sequence operably linked upstream of the nucleic acid sequence encoding the suppressor tRNA.
8. The method of claim 1 or 2 , wherein the inducible promoter of the suppressor construct is an arabinose BAD promoter.
9. The method of claim 8 , wherein the suppressor construct further comprises an araC regulatory gene.
10. The method of claim 1 or 2 , wherein the inducible promoter of the suppressor construct is an inducible T7 promoter.
11. The method of claim 1 , 2 or 3 , wherein the termination codon is UAG, UAA or UGA.
12. The method of claim 1 , 2 or 3 , wherein the suppressor tRNA is tRNA Ala , tRNA Glu , tRNA Phe or tRNA Pro .
13. The method of claim 1 , 2 or 3 , wherein the termination codon is UAG and the suppressor tRNA is tRNA Ala .