Display of antibody fragments on virus-like particles of rna bacteriophages

A nucleic acid construct comprising:

• (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of a bacteriophage coat protein or single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is optionally modified to contain a suppressible nonsense codon;
• (b) a nucleotide sequence which encodes an antibody single-chain variable fragment (scFv) and which is in-frame with, and positioned 3′ to, the coat polypeptide dimer coding sequence's termination codon;
• (c) a first restriction site positioned 3′ to the coat polypeptide dimer coding sequence and 5′ to the antibody single-chain variable fragment nucleotide sequence and a second restriction site positioned 3′ to the antibody single-chain variable fragment nucleotide sequence;
• (d) a PCR primer positioned 3′ to the second restriction site;
• (e) a gene for resistance to a first antibiotic;
• and
• (f) a replication origin for replication in a prokaryotic cell, and
• Optionally, a second plasmid, in combination with said first plasmid which comprises:
• (a) the gene for a nonsense suppressor tRNA to promote translational readthrough of the coat protein stop codon in said first plasmid, thus allowing the synthesis of a coat protein-scFv fusion protein;
• (b) a prokaryotic origin of replication;
• and
• (c) a gene for resistance to a second antibiotic.

A method for constructing a library of virus-like particles, the method comprising:

• (a) providing a plurality of a nucleic acid constructs comprising
• (1) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of a bacteriophage single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to contain a suppressible stop codon
• (2) a first restriction site positioned 3′ to the coat polypeptide dimer coding sequence and 5′ to the antibody single-chain variable fragment nucleotide sequence and a second restriction site positioned 3′ to the antibody single-chain variable fragment nucleotide sequence;
• (3) a gene for resistance to a first antibiotic, and
• (4) a replication origin for replication in a prokaryotic cell;
• (b) treating the nucleic acid constructs with a restriction enzyme;
• (c) obtaining a population of transcription units by inserting into the nucleic acid constructs, in a position which is in-frame with and 3′ to the coat polypeptide dimer coding sequence's termination codon, a nucleotide sequence which encodes an antibody single-chain variable fragment;
• and
• (d) expressing the transcription units and, optionally, isolating the library, wherein each particle comprises a bacteriophage coat polypeptide modified by insertion of the antibody single-chain variable fragment, and wherein the antibody single-chain variable fragment is displayed on the virus-like particle and encapsidates bacteriophage mRNA.

A method of expressing a library of scFv sequences displayed on VLPs, and affinity-selecting one or more VLP-scFv's with binding activity for a specific target molecule comprising:

• 1. Constructing a library of scFv's in an expression plasmid;
• 2. Introducing the library obtained in step 1 into an expression cell where each transformant produces a VLP displaying an scFv with a different ligand specificity;
• 3. Extracting the VLPs obtained in step 2 from the expression cells;
• 4. Subjecting the extracted VLPs to affinity selection of a binding target;
• 5. Washing away and discarding VLPs that fail to bind the binding target during step 4;
• 6. Eluting VLPs that bind the binding target;
• 7. Copying the RNA contained within the VLPs eluted from step 6 into DNA by reverse transcription and amplifying said DNA by polymerase chain reaction;
• and
• 8. Recloning the amplified DNA obtained from step 7 for production of VLPs, which VLPs are either used in additional rounds of affinity selection, or, if affinity selection is complete, characterizing the selected scFv's with respect to binding affinity and specificity and optionally, determining their sequences.