# US 10,119,159 B2 · Method of recombinant macromolecular production

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- Number: US 10,119,159 B2
- Document ID: US10119159B2
- Status: Granted
- Kind: B2
- Application number: 15/802,056
- Filed: 2017-11-02
- Published: 2018-04-26
- Granted: 2018-11-06
- Earliest claimed: 2010-09-26
- Assignees: Da Yu Enterprises, L.L.C.
- Applicants: Da Yu Enterprises, L.L.C.
- Inventors: Donald O'Keefe
- Web page: https://patentgrep.com/patent/US10119159B2

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12N 1/066: by physical processes
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 2500/10: involving cells
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods for in situ drug screening are provided which involve culturing a host cell containing two nucleic acid sequences, the first nucleic acid sequence encoding a membrane-permeabilizing agent and the second nucleic acid sequence encoding a desired macromolecule under the operative control of an inducible promoter, to a selected cell density to produce a macromolecule. Thereafter the host cells are exposed to an environmental condition that induces the agent to disrupt the integrity of the cell membrane without complete lysis of the cell membrane, thereby allowing transport through the membrane of small molecular weight compounds. These resulting host cells are provided with a nutrient cocktail containing components that can transport through the disrupted cell membrane, e.g., an inducing agent that induces the tightly regulated promoter and metabolic requirements that permit expression of the macromolecule. Bacterial host cells that can be used in these methods are also provided.

## Claims

### Claim 1

A method for in situ drug screening comprising:
(a) culturing multiple host cells, each of the cells containing:
i. a first nucleic acid sequence encoding a membrane-permeabilizing agent under the operative control of a first regulatory sequence that directs expression of the agent in the host cells; and
ii. a second nucleic acid sequence encoding a macromolecule under the operative control of a second regulatory sequence comprising an inducible promoter, the second regulatory sequence directing the expression of the macromolecule in the host cells;
to a selected cell density sufficient to produce a desired level of the macromolecule; wherein the second nucleic acid sequence in each of the host cells encodes a different macromolecule;
(b) exposing the host cells of (a) to an environmental condition that induces the membrane-permeabilizing agent to disrupt the integrity of the host cell membrane without complete lysis of the host cell membrane, cancelling the energized membrane function but allowing transport through the disrupted host cell membrane of small molecular weight compounds;
(c) providing the host cells with a nutrient cocktail comprising components that can transport through the disrupted host cell membrane, the components comprising an inducing agent that induces the promoter of the second regulatory sequence; metabolic requirements that permit expression of the macromolecule in the membrane-disrupted host cells and a chemical energy source; wherein the macromolecules are expressed in the host cells;
(d) distributing each of the host cells to separate wells of a mini-well plate after step (a), after step (b) or after step (c);
(e) treating the host cells of each well of step (d) with a test reagent; and
(f) determining the identity of the macromolecule that responded to the test reagent.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the response of the cells to a test reagent is a binding or triggering of a signal event.

### Claim 3 (depends on 1)

The method according to claim 1, further comprising lysing the cells prior to treating the cells with a test reagent.

### Claim 4 (depends on 1)

The method according to claim 1, wherein a test reagent is a small molecule that transports through the disrupted cell membranes.

### Claim 5

A method for in situ drug screening comprising:
(a) culturing host cells that comprise
(i) a first nucleic acid sequence encoding a membrane-permeabilizing agent under the operative control of a first regulatory sequence that directs expression of the agent in the host cells; and
(ii) a second nucleic acid sequence encoding a macromolecule under the operative control of a second regulatory sequence comprising an inducible promoter, the second regulatory sequence directing the expression of the macromolecule in the host cells,
to a selected cell density sufficient to meet a desired production level of the macromolecule;
(b) exposing the host cells of (a) to an environmental condition that induces the agent to disrupt the integrity of the host cell membrane without complete lysis of the host cell membrane, cancelling the energized membrane function but allowing transport through the disrupted host cell membrane of small molecular weight compounds;
(c) providing the host cells of (b) with a nutrient cocktail comprising components that can transport through the disrupted host cell membrane, the components comprising an inducing agent that induces the promoter of the second regulatory sequence of the cells and metabolic requirements that permit expression of the macromolecule in the membrane-disrupted host cells and a chemical energy source; wherein the macromolecule is expressed in the host cells;
(d) distributing the host cells to separate wells of a mini-well plate after step (a), after step (b) or after step (c);
(e) treating the host cells of (d) in each well with a different test reagent; and
(f) determining the identity of the test reagent that induced a response in the macromolecule.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the response of the cells to a test reagent is a binding or triggering of a signal event.

### Claim 7 (depends on 5)

The method according to claim 5, further comprising lysing the cells prior to treating the cells with a test reagent.

### Claim 8 (depends on 5)

The method according to claim 5, wherein a test reagent is a small molecule that transports through the disrupted cell membranes.

### Claim 9

A bacterial cell culture comprising a bacterial host cell comprising:
(i) a first nucleic acid sequence encoding a membrane-permeabilizing agent, under the operative control of a first regulatory sequence that directs expression of the membrane-permeabilizing agent in the host cell;
(ii) a second nucleic acid sequence encoding a macromolecule under the operative control of a second regulatory sequence comprising an inducible promoter, the second regulatory sequence directing the expression of the macromolecule in the host cell, and
(iii) a disrupted, incompletely lysed cell membrane with a cancelled energized membrane function,
said cell capable of allowing transport through the disrupted cell membrane of small molecular weight compounds and capable of expressing the macromolecule in the presence of a nutrient cocktail comprising an inducing agent that induces the promoter of the second regulatory sequence, metabolic requirements that permit expression of the macromolecule in the membrane-disrupted host cell, and a chemical energy source,
wherein said bacterial cell culture is at a cell density sufficient to meet the desired production levels of the macromolecule and wherein the macromolecule is expressed in the host cell.