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US 9,540,657 B2Granted
Expression of secreted and cell-surface polypeptides
AssigneeCalifornia Institute of Technology
InventorsKenneth Yu, David Baltimore, Lili Yang
Life of this patent
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Abstract
Some embodiments herein provide compositions and methods for expressing secreted and cell-surface-bound polypeptides in a single cell. In some embodiments, secreted and cell-surface polypeptide are produced from a single polynucleotide. The polynucleotide can comprise a sequence (or sequence encoding a polypeptide) that mediates separation of a membrane anchor from the polypeptide. In some embodiments, a desired ratio of secreted to surface-bound polypeptide is obtained by selecting a sequence that mediates a desired level of separation of the membrane anchor from the polypeptide.
Claims32
A polynucleotide construct comprising:
- a signal polynucleotide encoding a signal sequence sufficient to mediate translocation of a polypeptide comprising said signal sequence to a cell surface;
- a first cleavage polynucleotide encoding a first 2A polypeptide cleavage site in-frame with said signal sequence;
- an anchor polynucleotide encoding a membrane anchor polypeptide in-frame with said first 2A polypeptide cleavage site, wherein said membrane anchor polypeptide is sufficient to attach a polypeptide encoded by said polynucleotide construct to said cell surface, wherein at least one of (a) a majority of amino acids of said membrane anchor polypeptide are hydrophobic, or (b) said membrane anchor polypeptide facilitates the attachment of a fatty acid thereto;
- and
- a first insertion site for a first polypeptide-encoding polynucleotide encoding a first polypeptide,
- wherein said first insertion site is positioned for inserting a first polypeptide-encoding polynucleotide in-frame with said signal polynucleotide, said first cleavage polynucleotide, and said anchor polynucleotide,
- wherein said first insertion site is configured so that if said first polypeptide is expressed:
- (i) if said first 2A polypeptide cleavage site is cleaved, said first polypeptide is not attached to said membrane anchor polypeptide, and is thereby a secreted polypeptide;
- and (ii) if said first 2A polypeptide cleavage site is not cleaved, said first polypeptide comprises said membrane anchor polypeptide, and is thereby a membrane-bound polypeptide,
- wherein said first cleavage polynucleotide is positioned 3′ of said first insertion site, and wherein said first cleavage polynucleotide is positioned 5′ of a 3′ end of said anchor polynucleotide, and
- wherein said signal polynucleotide is 5′ of said first cleavage polynucleotide.
8 more elements
A method of expressing a secreted polypeptide and a surface-bound polypeptide from a single construct in a target cell, the method comprising:
- providing a construct comprising:
- a first polynucleotide encoding a first polypeptide,
- a signal polynucleotide encoding a signal sequence sufficient to mediate translocation of a polypeptide comprising said signal sequence to a surface of said target cell, wherein said signal sequence is in-frame with the first polynucleotide;
- a first cleavage polynucleotide encoding a first 2A polypeptide cleavage site in-frame with said signal sequence;
- and
- an anchor polynucleotide encoding a membrane anchor polypeptide in-frame with first 2A polypeptide cleavage site, wherein said membrane anchor polypeptide is sufficient to attach a polypeptide encoded by said construct to said surface of said target cell, wherein at least one of (a) a majority of amino acids of said membrane anchor polypeptide are hydrophobic, or (b) said membrane anchor polypeptide sequence facilitates the attachment of a fatty acid to said membrane anchor polypeptide,
- wherein said first polynucleotide is 5′ of said first cleavage polynucleotide,
- wherein said signal polynucleotide is 5′ of said first cleavage polynucleotide, and wherein said first cleavage polynucleotide is 5′ of said anchor polynucleotide;
- and
- delivering said construct to a target cell, wherein said target cell transcribes said construct, wherein once transcribed, said first cleavage site is cleaved in a first plurality of first polypeptides, so that said first plurality of first polypeptides do not comprise said membrane anchor polypeptide, and are thereby secreted by said target cell, and wherein said first cleavage site is not cleaved in a second plurality of first polypeptides, so that said second plurality of first polypeptides comprise said membrane anchor polypeptide, and are thereby bound to the surface of said target cell;
- and
- detecting a quantity of said second plurality of first polypeptides on the surface of said target cell.
9 more elements
Dependencies are inferred from the claim text.