Mammalian cell line for protein production and library generation

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...

AI Technical Summary

Problems solved by technology

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

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