Novartis Ag

Burkhard Wilms

Hans-Peter Knopf

Thomas Jostock

Zorica Dragic

Rita Schmitz

Audrey Nommay

Sabine Geisse

Audrey Josiane Nommay

The invention provides vector nucleic acid for expressing at least one polypeptide of interest in a mammalian cell, comprising

Mammalian expression vector

The present invention is based on the finding that the combination of a specific 5'UTR polynucleotide sequence (see SEQ ID NO 1) and the hCD33 secretory leader sequence in an expression cassette for expressing a polypeptide of interest results in a surprisingly better expression level of the polypeptide of interest compared to prior art expression cassettes. Based on this finding, the present invention inter alia provides novel expression cassettes, expression vectors and methods for producing a polypeptide of interest with high yield.

Optimized expression cassette for expressing a polypeptide with high yield

A method for selecting mammalian host cells that express a polypeptide of interest with high yield is described. The host cells comprise an expression cassette comprising at least a first polynucleotide 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. The host cells are cultivated 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 that is displayed on the cell surface. The cells are selected based upon the presence or amount of the displayed fusion polypeptide. Also provided are methods for producing a polypeptide that employ a respective selection to identify high expressing host cells.

Cell surface display of polypeptide isoforms by stop codon readthrough

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.

The invention provides vector nucleic acid for expressing at least one polypeptide of interest in a mammalian cell, comprising Also provided are host cells, comprising said vector and methods for producing a polypeptide using respective host cells.

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Weil am Rhein, Germany

Burkhard Wilms