# US 7,611,866 B2 · Selection of bacterial inner-membrane anchor polypeptides

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- Number: US 7,611,866 B2
- Document ID: US7611866B2
- Status: Granted
- Kind: B2
- Application number: 11/084,717
- Filed: 2005-03-18
- Published: 2005-11-24
- Granted: 2009-11-03
- Earliest claimed: 2002-07-15
- Assignees: Board of Regents, The University of Texas System
- Inventors: George Georgiou; Ki Jun Jeong; Barrett R. Harvey; Brent L. Iverson
- Web page: https://patentgrep.com/patent/US7611866B2

## 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
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C12N 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- C07K 2319/04: containing an ER retention signal such as a C-terminal HDEL motif
- 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)
- 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)

## Abstract

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating polypeptides capable of anchoring heterologous polypeptides to a bacterial inner membrane. In the technique, libraries of candidate anchor polypeptides are expressed as fusions with a heterologous polypeptide that is capable of being detected when bound to the inner membrane. In bacteria expressing a functional anchor sequence, the heterologous polypeptide becomes bound to outer face of the inner membrane. Bacteria with the functional anchor sequence can be identified by removing the outer membrane to remove non-anchored heterologous polypeptide followed by detection of anchored heterologous polypeptide. Such bacteria may be detected in numerous ways, including use of direct fluorescence or secondary antibodies that are fluorescently labeled, allowing use of efficient techniques such as fluorescence activated cell sorting (FACS).

## Claims

### Claim 1

A method of obtaining a bacterium comprising a nucleic acid sequence encoding an inner membrane anchor polypeptide that anchors a heterologous polypeptide to the outer side of the inner membrane of a Gram negative bacterium comprising the steps of:
(a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm; said bacterium comprising a nucleic acid sequence encoding a fusion between a heterologous polypeptide and a candidate inner membrane anchor sequence;
(b) removing the outer membrane; and
(c) selecting the bacterium based on the presence of the heterologous polypeptide anchored to the outer side of the inner membrane to identify an inner membrane anchor polypeptide that anchors a heterologous polypeptide to the outer side of the inner membrane of said bacterium.

### Claim 2 (depends on 1)

The method of claim 1, further defined as a method of obtaining a nucleic acid sequence encoding an inner membrane anchor sequence that anchors a heterologous polypeptide to the outer side of the inner membrane, the method further comprising the step of:
(d) cloning a nucleic acid sequence encoding the inner membrane anchor polypeptide.

### Claim 3 (depends on 1)

The method of claim 1, wherein selecting said bacterium comprises detecting the heterologous polypeptide with a binding polypeptide having specific affinity for the heterologous polypeptide.

### Claim 4 (depends on 3)

The method of claim 3, further comprising use of at least a second binding polypeptide having affinity for the heterologous polypeptide and/or the binding polypeptide having specific affinity for the heterologous polypeptide.

### Claim 5 (depends on 3)

The method of claim 3, wherein the second binding polypeptide is an antibody or fragment thereof.

### Claim 6 (depends on 5)

The method of claim 5, wherein the antibody or fragment thereof is fluorescently labeled.

### Claim 7 (depends on 3)

The method of claim 3, wherein selecting said bacterium comprises use of at least a third binding polypeptide having specific affinity for the heterologous polypeptide and/or said second binding polypeptide to label said bacterium.

### Claim 8 (depends on 1)

The method of claim 1, wherein the heterologous polypeptide comprises a detectable label.

### Claim 9 (depends on 8)

The method of claim 8, wherein the detectable label is an antigen.

### Claim 10 (depends on 8)

The method of claim 8, wherein the detectable label is GFP.

### Claim 11 (depends on 1)

The method of claim 1, wherein said heterologous polypeptide comprises an antibody or fragment thereof.

### Claim 12 (depends on 11)

The method of claim 11, wherein selecting the bacterium comprises detecting the antibody or fragment thereof with a labeled ligand having specific affinity for the antibody or fragment thereof.

### Claim 13 (depends on 1)

The method of claim 1, wherein said Gram negative bacterium is an E. coli bacterium.

### Claim 14 (depends on 1)

The method of claim 1, wherein step (a) is further defined as comprising providing a population of Gram negative bacteria.

### Claim 15 (depends on 14)

The method of claim 14, wherein said population of bacteria is further defined as collectively expressing a plurality of candidate inner membrane anchor sequences.

### Claim 16 (depends on 14)

The method of claim 14, wherein from about two to six rounds of selecting are carried out to obtain said bacterium from said population.

### Claim 17 (depends on 2)

The method of claim 2, wherein the bacterium is non-viable.

### Claim 18 (depends on 2)

The method of claim 2, wherein the bacterium is viable.

### Claim 19 (depends on 2)

The method of claim 2, wherein cloning comprises amplification of the nucleic acid sequence.

### Claim 20 (depends on 1)

The method of claim 1, wherein selecting is carried out by flow-cytometry or magnetic separation.

### Claim 21 (depends on 1)

The method of claim 1, wherein said nucleic acid encoding a candidate inner membrane anchor polypeptide is flanked by known PCR primer sites.

### Claim 22 (depends on 1)

The method of claim 1, wherein the candidate inner membrane anchor polypeptide is anchored to the outer side of the inner membrane with a transmembrane protein comprising the first six amino acids of NlpA.

### Claim 23 (depends on 22)

The method of claim 22, wherein the candidate inner membrane anchor polypeptide is anchored via an N- or C-terminus of the polypeptide.