Lambda phage display system and the process

A process of obtaining recombinant lambdoid bacteriophage with high density display of functional peptides and proteins on surface of said bacteriophage comprising the steps of:

• a) Constructing a donor plasmid comprising:
• A first recombination sequence, and
• a second recombination sequence, wherein said first and second recombination sequences are incompatible with each other, and wherein said first and second recombination sequences flank
• a nucleotide sequence that defines the elements for replication of the plasmid in bacteria,
• a nucleotide sequence encoding a first selectable marker, and
• a first inducible cistron comprising a lambdoid capsid/exogenous protein fusion protein encoding sequence) constructing a recipient phage having a nucleotide sequence comprising:
• lambdoid replication and packaging elements,
• said first recombination sequence, and
• said second recombination sequence, wherein said first and second recombination sequences flank
• a second inducible cistron for expression of a second selectable marker;
• c) transferring the polynucleotide sequences located between said first and second recombination sequences of said donor plasmid to said recipient phage to obtain cointegrates;
• d) propagating said cointegrates in liquid medium selective for said first selectable marker;
• and
• e) harvesting phages displaying said fusion protein,
• wherein said plasmid has the same genetic map as shown in FIG. 2 for pVCDcDL1.

A process of obtaining recombinant lambdoid bacteriophage with high density display of functional peptides and proteins on surface of said bacteriophage comprising the steps of:

• a) Constructing a donor plasmid comprising:
• A first recombination sequence, and
• a second recombination sequence, wherein said first and second recombination sequences are incompatible with each other, and wherein said first and second recombination sequences flank
• a nucleotide sequence that defines the elements for replication of the plasmid in bacteria,
• a nucleotide sequence encoding a first selectable marker, and
• a first inducible cistron comprising a lambdoid capsid/heterologous polypeptide fusion protein encoding sequence;
• b) constructing a recipient phage having a nucleotide sequence comprising:
• lambdoid replication and packaging elements,
• said first recombination sequence, and
• said second recombination sequence, wherein said first and second recombination sequences flank
• a second inducible cistron for expression of a second selectable marker;
• c) transferring the polynucleotide sequences located between said first and second recombination sequences of said donor plasmid to said recipient phage to obtain cointegrates;
• d) propagating said cointegrates in liquid medium selective for said first selectable marker;
• and
• e) harvesting phages displaying said fusion protein,
• wherein said cointegrate containing recombinant phage particles comprise a matrix of proteins encapsulating a lambdoid genome encoding said fusion protein,
• wherein said fusion protein is surface accessible in said matrix, and
• wherein said fusion protein comprises gpD lambdoid bacteriophage capsid polypeptide, a linker polypeptide, a protease cleavage site, a linker poly peptide, and said heterologous polypeptide.

A process of obtaining recombinant lambdoid bacteriophage with high density display of functional peptides and proteins on surface of said bacteriophage comprising the steps of:

• a) Constructing a donor plasmid comprising:
• A first recombination sequence, and
• a second recombination sequence, wherein said first and second recombination sequences are incompatible with each other, and wherein said first and second recombination sequences flank
• a nucleotide sequence that defines the elements for replication of the vector in bacteria,
• a nucleotide sequence encoding a first selectable marker, and
• a first inducible cistron comprising a lambdoid capsid/heterologous polypeptide fusion protein encoding sequence;
• b) constructing a recipient phage having a nucleotide sequence comprising:
• a lambdoid replication and packaging elements,
• said first recombination sequence, and
• said second recombination sequence, wherein said first and second recombination sequences flank
• a second inducible cistron for expression of a second selectable marker
• c) transferring the polynucleotide sequences located between said first and second recombination sequences of said donor plasmid to said recipient phage to obtain cointegrates;
• d) propagating said cointegrates in liquid medium selective for said first selectable marker;
• and
• e) harvesting phages displaying said fusion protein,
• wherein the number of fusion protein molecules on the surface of each recombinant lambdoid phage particle is 100-1000 fold more than capsid protein molecules on surface of each particle of filamentous phage M13.