Plasmids and methods for peptide display and affinity-selection on virus-like particles of rna bacteriophages

An isolated transcription unit comprising:

• a bacterial or bacteriophage promoter;
• a coding sequence of an RNA bacteriophage single chain coat polypeptide dimer comprising an upstream subunit and a downstream subunit, a site for insertion of a heterologous peptide located in the downstream subunit;
• a stop codon replacing a codon which encodes a first amino acid of the downstream subunit of said coat protein;
• and,
• a bacterial or bacteriophage terminator.

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

• (a) providing a plurality of a nucleic acid constructs of claim 47;
• (b) treating the nucleic acid constructs with a restriction enzyme;
• (c) inserting coding sequences for a heterologous peptide into the nucleic acid constructs to obtain a population of transcription units;
• and
• (d) expressing the transcription units and, optionally, isolating the library, wherein each particle comprises a coat polypeptide of MS2 modified by insertion of a heterologous peptide, and wherein the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 mRNA.

A method for identifying a peptide having a property of interest, the method comprising:

• (a) providing a population of the virus-like particles of claim 19, wherein
• (1) each particle comprises a coat polypeptide of MS2 modified by insertion of a heterologous peptide, and wherein
• (2) the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 mRNA;
• (b) assaying heterologous peptides expressed on the virus-like particles for the property of interest.

A nucleic acid construct comprising:

• (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of bacteriophage MS2 or PP7 single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to (1) define a first restriction site which is located in the downstream portion of the coat polypeptide dimer coding sequence and which is either positioned 5′ to, or located within, the sequence which defines the coat polypeptide dimer AB loop, and (2) to contain a nucleotide sequence (NNS)x, where N is any nucleotide, S is a guanosine nucleotide (G) or cytidine nucleotide (C), and x is an integer from 1 to 500;
• (b) a second restriction site positioned 3′ to the coat polypeptide dimer coding sequence;
• (c) PCR primers positioned 5′ to the first restriction site and 3′ to the second restriction site;
• (d) an antibiotic resistance gene;
• and
• (e) a replication origin for replication in a prokaryotic cell.

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

• (a) providing a plurality of a nucleic acid constructs according to claim 116;
• (b) treating the nucleic acid constructs with a restriction enzyme;
• (c) inserting coding sequences for a heterologous peptide into the nucleic acid constructs to obtain a population of transcription units;
• and
• (d) expressing the transcription units and, optionally, isolating the library, wherein
• each particle comprises a coat polypeptide of MS2 or PP7 modified by insertion of a heterologous peptide, and wherein the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 or PP7 mRNA, respectively.

A method for affinity selection of vaccine candidates, the method comprising:

• (a) constructing a population of virus-like particles (VLPs) displaying a random sequence or antigen fragment library of peptides, wherein each particle comprises a single chain dimer of a coat polypeptide of MS2 or PP7 modified by insertion of a heterologous peptide from said library of peptides, and wherein (2) the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 or PP7 mRNA, respectively;
• (b) incubating the population of VLPs from step (a) to an immobilized monoclonal or polyclonal antibody to allow binding of said VLPs to said antibody;
• (c) washing away unbound VLPs and eluting bound VLPs from said antibody;
• (d) denaturing said eluted VLPs to obtain RNA contained therein and copying said RNA into DNA and amplifying said DNA by PCR;
• (e) obtaining the amplified DNA and recloning said DNA into a plasmid expression vector;
• (f) introducing said expression vector into an expression cell to produce a population of VLPs and isolating said VLPs from said expression cells.