Mammalian cell line for protein production and library generation

A mammalian and cell line technology, applied in the field of protein libraries for directed evolution and protein engineering, can solve problems such as ineffective expression and screening of proteins

Pending Publication Date: 2018-12-21
ETH ZZURICH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Critically, however, standard systems cannot efficiently express and screen some complex proteins (e.g., full-length antibodies)

Method used

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  • Mammalian cell line for protein production and library generation
  • Mammalian cell line for protein production and library generation
  • Mammalian cell line for protein production and library generation

Examples

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Embodiment Construction

[0110] The present invention is further illustrated by the following examples and drawings, from which further embodiments and advantages can be obtained. These examples are intended to illustrate the invention without limiting its scope.

[0111] To carry out the present invention, the inventors have generated a plug-and-(dis)play (PnP) mammalian cell line. The inventors used mouse B lymphocytes (hybridoma cells), which serve as production factories for antibody proteins. PnP cell lines consist of the following components:

[0112] 1) Endogenous V in the IgH locus H The gene was replaced by a fluorescent protein (mRuby, originally called mRuby2, derived from Addgene.org plasmid #:40260 (Lam et al., Nat Methods 2012, 9:1005-1012)). This was achieved by transfecting WT hybridoma cells with a CRISPR-Cas9 plasmid (pX458) (Addgene.org plasmid #: 48138) (Cong et al., Science 2013, 339:819-823) with a guide RNA (gRNA), so The guide RNA targets V H and the intron between the IgG...

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Abstract

According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

Description

[0001] The present invention relates to engineered cell lines and their use for the rapid generation of stable cells for protein expression and the generation of protein libraries for directed evolution and protein engineering. Background technique [0002] Currently available methods for protein production from mammalian cells rely primarily on random transgene integration in three cell lines: human embryonic kidney 293 (HEK293) cells, mouse myeloma cells (Sp20 and NS0), and Chinese hamster ovary ( CHO) cells. These methods are expensive and time-consuming. The biotechnology industry standard for therapeutic protein production calls for the generation of stable recombinant protein producing cell lines capable of nearly unlimited protein production when recovered from frozen cell stocks. [0003] Engineering proteins by directed evolution requires in vitro mutagenesis methods (e.g., error-prone PCR, degenerate primer PCR mutagenesis, DNA shuffling), followed by cloning into e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N15/64C12N15/90
CPCC12N5/0635C12N15/102C12N15/1037C12N15/907C12N2510/00C12N2510/02C12Q2521/539C12N2800/80C12N15/1034C12N9/22C12N15/1058C40B20/04C12N15/1082C40B40/08C12N15/113C12N15/1086C12N2310/20C40B10/00
Inventor S·雷迪W·凯尔顿C·帕罗拉D·梅森M·博格森
Owner ETH ZZURICH
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