Expression of secreted and cell-surface polypeptides

摘要

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.

主权项

1. 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.