# US 7,410,801 B2 · Lambda phage display system and the process

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- Number: US 7,410,801 B2
- Document ID: US7410801B2
- Status: Granted
- Kind: B2
- Application number: 10/515,150
- Filed: 2003-05-20
- Published: 2005-08-18
- Granted: 2008-08-12
- Earliest claimed: 2002-05-20
- Assignees: University of Delhi Department of Biochemistry
- Inventors: Vijay Kumar Chaudhary; Amita Gupta; Sankar Adhya; Ira Pastan
- Web page: https://patentgrep.com/patent/US7410801B2

## CPC

- C40B 40/02: Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a process of obtaining recombinant lambdoid bacteriophage with high density display of functional peptides and proteins on surface of said phage comprising of: constructing a donor plasmid having a nucleotide sequence that defines the elements for replication of the vector in bacteria, a selectable marker, a nucleotide sequence flanked by two non-compatible recombination sequences, and an inducible cistron for expression of a capsid protein and a fusion protein; constructing a recipient phage having a nucleotide sequence that defines the lambdoid elements for replication and packaging of the vector into an assembled bacteriophage and encodes an inducible cistron for expression of a selectable marker flanked by two non-compatible recombination sequences; transferring the said donor plasmid to said recipient plasmid to obtain cointegrates; growing said cointegrates in selective liquid medium; harvesting phages displaying protein encoded by the foreign DNA encapsulated in said harvested phage particle.

## Claims

### Claim 1

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 promoter for transcribing said first cistron,
a first ribosome binding site,
a first translatable nucleotide sequence encoding a lambdoid bacteriophage capsid polypeptide
a second translatable sequence operatively linked to said first translatable sequence and encoding a first linker polypeptide, a protease cleavable peptide sequence, a second linker polypeptide, and
a sequence adapted for ligation of an insert polynucleotide or an MCS;
wherein each translatable nucleotide sequence is in the same reading frame such that induction of said first cistron expresses a fusion polypeptide consisting of said capsid polypeptide, linker polypeptide, protease cleavage site, linker polypeptide, and said MCS-encoded or said insert-encoded polypeptide;
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 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 polypeptide.

### Claim 2

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.

### Claim 3

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.

### Claim 4 (depends on 1)

A process as claimed in claim 1 wherein said heterologous polypeptide of the cointegrate-containing recombinant phage particles is selected from the group consisting of capsid protein p24 of Human Immunodeficiency Virus, decapeptide cmyc, and a single chain antibody fragment (scFv).

### Claim 5 (depends on 2)

A process as claimed in claim 2 wherein said heterologous polypeptide of the cointegrate-containing recombinant phage particles is capsid protein p24 of Human Immunodeficiency Virus.

### Claim 6 (depends on 2)

A process as claimed in claim 2 wherein said heterologous polypeptide of the cointegrate-containing recombinant phage particles is decapeptide cmyc.

### Claim 7

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.