Recombinant mucins, and compositions and methods for using the same
Provided are compositions and methods related to improved mucins, methods of making the improved mucins, and cells and cell cultures that express glycosylated mucins. The compositions and methods provide improved cell cultures, and improved methods of producing co-expressed proteins that are distinct from the mucins.
1 . Modified mammalian cells present in a suspension cell culture, said culture being present in a suspended cell bioreactor, the modified mammalian cells comprising polypeptides expressed from recombinant polynucleotides introduced into the cells, wherein the polypeptides comprise a transmembrane anchor and a segment external to the cells, the segment external to the cells comprising a repeated amino acid sequence consisting of the sequence PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 8), said sequence being repeated only 21 or 42 times, and wherein the cells in the suspension cell culture exhibit less aggregation relative to a control value obtained from a suspended cell culture comprising mammalian cells that do not express the polypeptide comprising the repeated amino acid sequence.
2 . The modified mammalian cells of claim 1 , wherein the cells are human cells.
3 . The modified mammalian cells of claim 2 , wherein the cells are human embryonic kidney cells.
4 . The modified mammalian cells of claim 3 , wherein the modified mammalian cells further comprise an introduced polynucleotide encoding a distinct polypeptide that is different from the polypeptides comprising the repeated amino acid sequence, and wherein the modified mammalian cells are capable of producing the distinct polypeptide.
5 . The modified mammalian cells of claim 3 , wherein the transmembrane anchor comprises a cytoplasmic recycling motif.
6 . The modified mammalian cells of claim 1 , wherein the recombinant polynucleotides encoding the segment external to the cells that comprises the repeated amino acid sequence consisting of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8) repeated only 21 times comprise a sequence consisting of SEQ ID NO:51.
7 . The modified mammalian cells of claim 1 , wherein the recombinant polynucleotides encoding the segment external to the cells that comprises the repeated amino acid sequence consisting of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:8) repeated only 42 times comprise a sequence consisting of SEQ ID NO:50.
8 . A cell suspension bioreactor comprising a suspension cell culture comprising modified mammalian cells according to claim 3 .
9 . An isolated polynucleotide encoding the polypeptides comprising a transmembrane anchor and a repeated amino acid sequence according to claim 1 .
10 . The isolated polynucleotide of claim 9 , wherein the isolated polynucleotide is present in an expression vector.
11 . A method of making cells that express the polypeptides according to claim 1 , comprising introducing an isolated polynucleotide encoding the polypeptides into the cells such that the polypeptide is expressed.
12 . A method for producing a desired polypeptide, the method comprising expressing the desired polypeptide in the modified mammalian cells according to claim 1 , such that the desired polypeptide is produced, wherein the desired polypeptide is distinct from the polypeptides comprising the repeated amino acid sequence.
13 . The method of claim 12 , further comprising separating the desired polypeptide from the suspension cell culture.
14 . The method of claim 13 , wherein the modified mammalian cells are adapted to growth in a suspension culture.