Tricistronic vectors and uses therefor
A tricistronic vector (i.e., a vector capable of expressing three exogenous genes, which are not fused together, under the control of one promoter) effectively can encode an immunoglobulin-presenting polypeptide and two immunoglobulin (Ig) polypeptides. The encoded Ig-presenting polypeptide is able to associate with at least one of the Ig polypeptides via co-expressed associating agents. A vector according to the present invention particularly is suited for phage display technology, e.g., when the Ig-presenting polypeptide is a phage coat protein and the Ig polypeptides associate to form a Fab.
1. A library of tricistronic prokaryotic display vector constructs comprising:
a regulatable prokaryotic promoter;
a first nucleic acid sequence encoding a gIIp phage coat protein or C-terminal domain thereof;
a second nucleic acid sequence encoding a first immunoglobulin (Ig) polypeptide;
a third nucleic acid sequence encoding a second immunoglobulin (Ig) polypeptide;
a nucleic acid sequence encoding a first associating agent fused to or comprised within said nucleic acid sequence encoding the gIIp phage coat protein or C-terminal domain thereof, wherein said first associating agent comprises a cysteine residue; and
a nucleic acid sequence encoding a second associating agent fused to or comprised within said nucleic acid encoding the first immunoglobulin (Ig) polypeptide, wherein said second associating agent comprises a cysteine residue,
wherein said first, second and third nucleic acid sequences are under the control of said promoter, and wherein upon expression of said tricistronic vector, (i) said gIIIp phage coat protein or C-terminal domain thereof and said first Ig polypeptide associate via their respective associating agents and (ii) said first and second Ig polypeptides self-associate.
2. The library of vector constructs according to claim 1 , wherein said first and second Ig polypeptides self-associate to form a Fab or other functional Ig fragment.
3. The library of vector constructs according to claim 1 , wherein the first and second Ig polypeptides self-associate via non-covalent interactions.
4. The library of vector constructs according to claim 1 , further comprising a first secretory signal sequence in the same reading frame as the nucleic acid sequence encoding the first Ig polypeptide.
5. The library of vector constructs according to claim 4 , further comprising a second secretory signal sequence in the same reading frame as the nucleic acid sequence encoding the second Ig polypeptide.
6. The library of vector constructs according to claim 5 , further comprising a third secretory signal sequence in the same reading frame as the nucleic acid sequence encoding the phage coat protein or C-terminal domain thereof.
7. The library of vector constructs according to claim 1 , wherein said vector is a phagemid vector.
8. The library of vector constructs according to claim 1 , wherein the associating agents become disassociated in solution upon the addition of a reducing agent.
9. The library of vector constructs according to claim 1 , wherein said second associating agent is fused to said first Ig polypeptide via a peptide linker.
10. The library of vector constructs according to claim 6 , wherein said first, second, and third secretory signal sequences are prokaryotic signal sequences.
11. The library of vector constructs according to claim 1 , further comprising a ribosome binding site positioned 5-primeward of the nucleic acid sequence encoding the second Ig polypeptide.
12. The library of vector constructs according to claim 11 , further comprising a ribosome binding site positioned 5-primeward of the nucleic acid sequence encoding the first Ig polypeptide.
13. The library of vector constructs according to claim 12 , further comprising a ribosome binding site positioned 5-primeward of the nucleic acid sequence encoding the phage coat protein or C-terminal domain thereof.