# US 10,870,846 B2 · Cellular high throughput encapsulation for screening or selection > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,870,846 B2 - Document ID: US10870846B2 - Status: Granted - Kind: B2 - Application number: 14/371,031 - Filed: 2013-01-09 - Published: 2015-01-29 - Granted: 2020-12-22 - Earliest claimed: 2012-01-09 - Assignees: UniversitäT ZüRich - Applicants: Universität Zürich - Inventors: Daniel Scott; Andreas Plückthun - Web page: https://patentgrep.com/patent/US10870846B2 ## CPC - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms} - C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis} - 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) ## Abstract The invention relates to a method for selecting a sequence set from a library of expressed nucleic acid sequences, wherein cells are provided, each cell comprises an expressed nucleic acid sequence expressed as a target protein. The cells are encapsulated by treating them with a cationic polysaccharide and subsequently treating them with an anionic polysaccharide, yielding encapsulated cells, perforating the membrane of the encapsulated cells, yielding solubilized compartments, contacting them with a ligand to said target protein, the ligand bearing a detectable label, and selecting a subset of solubilized compartments as a function of detectable label and isolating the expressed nucleic acid sequences from the selection as a selected sequence set. ## Claims ### Claim 1 A method for selecting a sequence set from a library of expressed nucleic acid sequences, comprising: providing a plurality of cells, each cell comprising an expressed nucleic acid sequence expressed as a membrane protein in said cell, encapsulating said plurality of cells in a single encapsulation step, comprising: treating said plurality of cells with a cationic polysaccharide in a cationic treatment step, and treating said plurality of cells with an anionic polysaccharide in an anionic treatment step, wherein said encapsulating step gives rise to a plurality of encapsulated cells which each have a detergent resistant coating, and wherein each encapsulated cell comprises an expressed nucleic acid sequence expressed as the membrane protein, solubilizing after the single encapsulation step a membrane of said plurality of encapsulated cells with a detergent to form a plurality of solubilized compartments that retain detergent solubilized membrane protein, wherein the detergent resistant coating comprises pores that retain the detergent solubilized membrane protein, and wherein the nucleic acid sequence is not expressed in each of the plurality of solubilized compartments, labeling said plurality of solubilized compartments by contacting said plurality of solubilized compartments, in a labeling step, with a ligand that binds to said membrane protein, said ligand bearing a detectable label, or with an indicator of an enzymatic activity of said membrane protein, said enzymatic activity converting said indicator to a detectable label, wherein said labeling step gives rise to plurality of labeled solubilized compartments, selecting a subset of said plurality of labeled solubilized compartments as a function of detectable label present in said labeled solubilized compartments, and isolating said expressed nucleic acid sequences from the subset as a selected sequence set. ### Claim 2 (depends on 1) The method according to claim 1, wherein the cationic polysaccharide is chitosan. ### Claim 3 (depends on 1) The method according to claim 1, wherein the anionic polysaccharide is alginate or hyaluronic acid. ### Claim 4 (depends on 1) The method according to claim 1, wherein the cationic treatment step precedes the anionic treatment step. ### Claim 5 (depends on 1) The method according to claim 1, wherein a sequence of steps comprising a cationic treatment step followed by an anionic treatment step is repeated two to ten times. ### Claim 6 (depends on 1) The method according to claim 1, wherein the detectable label is a fluorescent dye. ### Claim 7 (depends on 1) The method according to claim 1, wherein said ligand is an oligopeptide, agonist, antagonist, substrate or transition state analogue binding to a variant of said membrane protein. ### Claim 8 (depends on 1) The method according to claim 1, wherein the selected sequence set is transferred into a plurality of cells and submitted to a sequence of encapsulation step, solubilization step, labelling step, selection step and isolation step. ### Claim 9 (depends on 1) The method according to claim 1, wherein after said isolation step, said selected sequence set is diversified by a. amplification of said expressed nucleic acid sequences by a process introducing mutations into the amplified sequence, and/or by b. deletion or insertion of sequence tracts into said expressed nucleic acid sequences, and subsequently, the selected sequence set is submitted to another sequence of encapsulation step, solubilization step, labelling step, selection step and isolation step. ### Claim 10 (depends on 1) The method according to claim 1, wherein said library of expressed nucleic acid sequences is a library of homologous sequences of at least 60% identity with each other. ### Claim 11 (depends on 1) The method according to claim 1, wherein said expressed nucleic acid sequence is comprised in a transgene expression construct. ### Claim 12 (depends on 11) The method according to claim 11, wherein said transgene expression construct is plasmid. ### Claim 13 (depends on 1) The method according to claim 1, wherein said membrane protein is a G-protein coupled receptor protein, an ion channel, or an enzyme. ### Claim 14 (depends on 1) The method according to claim 1, wherein said cells are bacterial cells. ### Claim 15 (depends on 1) The method according to claim 1, wherein said detectable label is a fluorescent dye and said selection step is accomplished by fluorescent cell sorting.