Cell surface display of polypeptide isoforms by stop codon readthrough

Patent metadata

Field	Value
Publication number	US-9758779-B2
Application number	US-200913060974-A
Country	US
Kind code	B2
Filing date	Aug 28, 2009
Priority date	Aug 28, 2008
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

The application describes a method of selecting mammalian host cells that express a polypeptide of interest with high yield. The host cells contain an expression cassette with a first polynucleotide encoding a polypeptide of interest, at least one leaky stop codon located downstream of the first polynucleotide and a second polynucleotide located downstream of the leaky codon encoding an immunoglobulin transmembrane anchor comprising a cytoplasmic domain. The host cells are cultivated to allow expression of the polypeptide of interest such that some of the polypeptides of interest are expressed as fusion proteins displayed on the cell surface. High producing cells are then selected based on the presence or amount of the displayed fusion polypeptides.

First claim

The invention claimed is:

1. A method for producing a polypeptide of interest with high yield, the method comprising:
a) providing a plurality of mammalian host cells comprising a heterologous nucleic acid comprising at least one cassette (Cas-POI) comprising a first polynucleotide (Pn-POI) encoding the polypeptide of interest, at least one leaky stop codon downstream of the first polynucleotide, and a second polynucleotide downstream of the leaky stop codon encoding an immunoglobulin transmembrane anchor comprising a cytoplasmic domain;
b) cultivating the mammalian host cells to allow expression of the polypeptide of interest such that at least a portion of the polypeptide of interest is expressed as a fusion polypeptide comprising the immunoglobulin transmembrane anchor directly anchored to the cell membrane comprising a cytoplasmic domain, wherein at least a portion of said fusion polypeptide comprising the immunoglobulin transmembrane anchor is displayed on the surface of said mammalian host cells to allow binding of a detection compound;
c) selecting at least one of the plurality of mammalian host cells based upon to the presence or amount of the fusion polypeptide displayed on the cell surface;
d) culturing the selected mammalian host cell in culture medium under conditions that allow for expression of the polypeptide of interest wherein the polypeptide of interest is secreted into the culture medium; and
e) obtaining the polypeptide of interest from the culture medium.

2. The method according to claim 1 , wherein the immunoglobulin transmembrane anchor is selected from the group consisting of
a) a transmembrane anchor derived from IgA, IgE, IgM, IgG and/or IgD, and
b) an immunoglobulin transmembrane anchor comprising a sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 7.

3. The method according to claim 1 , wherein step c) comprises contacting the plurality of mammalian host cells with a detection compound binding the fusion polypeptide and selecting at least one mammalian host cell based upon the presence or amount of the bound detection compound.

4. The method according to claim 1 , wherein two or more selection cycles are performed, wherein in each selection cycle at least one mammalian host cell is selected based upon the presence or amount of the fusion polypeptide displayed on the cell surface.

5. The method according to claim 3 , wherein binding of the detection compound to the surface of the mammalian host cell is detected by flow cytometry.

6. A method for producing a polypeptide of interest, comprising culturing a mammalian host cell which comprises at least one cassette comprising:
a) at least a first polynucleotide encoding a polypeptide of interest;
b) at least one leaky stop codon downstream of the first polynucleotide; and
c) a second polynucleotide downstream of the leaky stop codon encoding an immunoglobulin transmembrane anchor comprising a cytoplasmic domain,
wherein at least a portion of said immunoglobulin transmembrane anchor is displayed on the surface of said mammalian host cells to allow binding of an immunoglobulin.

7. The method for producing a polypeptide of interest according to claim 1 , further comprising one or both of the following steps:
a) purifying the expressed polypeptide; and
b) further processing or modifying the expressed polypeptide.

8. The method for producing a polypeptide of interest according to claim 6 , further comprising at least one step selected from the steps:
obtaining the polypeptide from the cell culture;
obtaining the polypeptide from the culture medium wherein the polypeptide is secreted into the culture medium;
isolating the expressed polypeptide;
purifying the expressed polypeptide; and
further processing or modifying the expressed polypeptide.

Assignees

• Jostock Thomas
• Knopf Hans-Peter
• Wilms Burkhard
• Nommay Audrey
• Novartis Ag

Inventors

• Jostock Thomas
• Knopf Hans-Peter
• Wilms Burkhard
• Nommay Audrey

Classifications

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C07K2319/035

  containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins · CPC title

• C12Q1/02

  involving viable microorganisms · CPC title

• C12N15/79 Primary

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title

• C07K19/00

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title