# US 11,306,318 B2 · Vector construct

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- Number: US 11,306,318 B2
- Document ID: US11306318B2
- Status: Granted
- Kind: B2
- Application number: 16/760,111
- Filed: 2018-11-05
- Published: 2020-08-13
- Granted: 2022-04-19
- Earliest claimed: 2017-11-03
- Assignees: Nextera AS
- Applicants: Nextera AS
- Inventors: Geir Åge Løset; Terje Frigstad; Sebastian Berge-Seidl; Nicolay Rustad Nilssen; Inger Sandlie
- Web page: https://patentgrep.com/patent/US11306318B2

## CPC

- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C07K 14/005: from viruses
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C07K 2319/43: containing a FLAG-tag
- C12N 2795/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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)

## Abstract

The present invention provides a vector construct comprising the following components: (i) a sequence encoding a signal peptide which directs proteins into the Tat secretory pathway; and (ii) a sequence encoding a fluorophore fused to a sequence encoding a pVIII phage coat protein. Nucleic acid molecules comprising components (i) and (ii) are also provided, together with phage particles comprising such vectors or nucleic acid molecules and expressing a fluorophore-pVIII fusion protein on the surface. Methods for producing such fluorescent phage particles are also provided.

## Claims

### Claim 1

A vector construct comprising:
(i) a polynucleotide sequence encoding a signal peptide which directs proteins into the Tat secretory pathway; and
(ii) a polynucleotide sequence encoding a fluorophore fused to a polynucleotide sequence encoding a pVIII phage coat protein.

### Claim 2 (depends on 1)

The vector construct of claim 1, wherein the fluorophore is a fluorophore having or comprising a β-barrel structure or architecture.

### Claim 3 (depends on 2)

The vector construct of claim 2, wherein the fluorophore is green fluorescent protein (GFP), a GFP-derivative, or has GFP architecture.

### Claim 4 (depends on 3)

The vector construct of claim 3, wherein the fluorophore is mNeonGreen comprising SEQ ID NO:4 or a variant sequence thereof having at least 70% identity to SEQ ID NO:4.

### Claim 5 (depends on 1)

The vector construct of claim 1, wherein the signal peptide is TorA comprising SEQ ID NO:32 or a variant sequence thereof having at least 70% identity to SEQ ID NO:32.

### Claim 6 (depends on 1)

The vector construct of claim 1, wherein the signal peptide is Tor AB7 comprising SEQ ID NO:2 or a variant sequence thereof having at least 70% identity to SEQ ID NO:2.

### Claim 7 (depends on 1)

The vector construct of claim 1, wherein the pVIII phage coat protein corresponds to the pVIII coat protein from M13, fd or f1 phage or a variant thereof.

### Claim 8 (depends on 1)

The vector construct of claim 1, wherein the pVIII phage coat protein comprises SEQ ID NO:8, or a variant sequence thereof having at least 70% identity to SEQ ID NO:8.

### Claim 9 (depends on 8)

The vector construct of claim 8, wherein the variant sequence has a valine to isoleucine mutation at position 33 of SEQ ID NO:8 or a corresponding position.

### Claim 10 (depends on 9)

The vector construct of claim 9, wherein the nucleic acid sequence encoding said isoleucine residue comprises the codon ATA.

### Claim 11 (depends on 9)

The vector construct of claim 9, wherein the pVIII phage coat protein comprises SEQ ID NO:61, or a variant sequence thereof having at least 70% identity to SEQ ID NO:61, or is encoded by a nucleic acid sequence comprising SEQ ID NO:60 or a variant sequence thereof having at least 70% identity to SEQ ID NO:60.

### Claim 12 (depends on 1)

The vector construct of claim 1, wherein the pVIII phage coat protein comprises SEQ ID NO:25, or a variant sequence thereof having at least 70% identity to SEQ ID NO:25.

### Claim 13 (depends on 1)

The vector construct of claim 1, wherein the vector further comprises a polynucleotide sequence encoding a linker between the fluorophore and the pVIII phage coat protein.

### Claim 14 (depends on 13)

The vector construct of claim 13, wherein the linker comprises SEQ ID NO:6 or a variant sequence thereof having at least 70% identity to SEQ ID NO:6.

### Claim 15 (depends on 1)

The vector construct of claim 1, wherein the vector further comprises a polynucleotide sequence encoding a detectable tag.

### Claim 16 (depends on 15)

The vector construct of claim 15, wherein the tag is located between the signal peptide and the fluorophore.

### Claim 17 (depends on 15)

The vector construct of claim 15, wherein the tag is a FLAG tag or a derivative thereof, or another negatively charged tag.

### Claim 18 (depends on 1)

The vector construct of claim 1, wherein the vector further comprises one or more ribosome binding sites (RBS).

### Claim 19 (depends on 18)

The vector construct of claim 18, wherein the RBS driving translation of the fluorophore-pVIII fusion protein is a weak RBS.

### Claim 20 (depends on 1)

The vector construct of claim 1, wherein the vector construct encodes a polypeptide sequence comprising SEQ ID NO:16: or a variant sequence thereof having at least 70% identity to SEQ ID NO:16.

### Claim 21 (depends on 1)

The vector construct of claim 1, wherein the vector construct encodes a polypeptide sequence comprising: (i) SEQ ID NO:18 or a variant sequence thereof having at least 70% identity to SEQ ID NO:18; (ii) SEQ ID NO:20 or a variant sequence thereof having at least 70% identity to SEQ ID NO:20; or (iii) SEQ ID NO:22 or a variant sequence thereof having at least 70% identity to SEQ ID NO:22.

### Claim 22 (depends on 21)

The vector construct of claim 21, wherein the pVIII phage coat protein comprises a variant sequence having a valine to isoleucine mutation at the position corresponding to position 33 of SEQ ID NO:8.

### Claim 23 (depends on 22)

The vector construct of claim 22, wherein the nucleic acid sequence encoding said isoleucine residue comprises the codon ATA.

### Claim 24 (depends on 1)

The vector construct of claim 1, wherein the vector construct further comprises a polynucleotide sequence encoding a protein of interest fused to a polynucleotide sequence encoding a non-pVIII phage coat protein.

### Claim 25 (depends on 1)

The vector construct of claim 1, wherein the vector is a phagemid or a phage vector.

### Claim 26 (depends on 9)

A nucleic acid molecule comprising a sequence encoding the pVIII phage coat protein variant sequence as defined in claim 9.

### Claim 27 (depends on 1)

Phage particles comprising the vector construct of claim 1 and expressing a fluorophore-pVIII fusion protein on the surface.

### Claim 28 (depends on 27)

The phage particles of claim 27, wherein said phage particles are particles of a filamentous phage.

### Claim 29 (depends on 27)

A library of phage particles, wherein the phage particles are as defined in claim 27, and wherein multiple different proteins of interest are expressed on the surface of the phage particles.

### Claim 30 (depends on 1)

A phage display system comprising a vector construct as defined in claim 1.

### Claim 31 (depends on 30)

The phage display system of claim 30, further comprising an E. coli host cell which over expresses the proteins Tat A, Tat B and Tat C.

### Claim 32 (depends on 1)

A method for producing fluorescent phage particles comprising:
introducing the vector construct as defined in claim 1 into a bacterial host cell.

### Claim 33 (depends on 32)

The method according to claim 32, wherein the bacterial host cell is an E. coli host cell.

### Claim 34 (depends on 33)

The method according to claim 33, wherein the E. coli host cell over expresses the proteins Tat A, Tat B and Tat C.