Membrane bound reporter molecules and their use in cell sorting

Abstract

The present invention relates to nucleic acid molecules comprising a nucleic acid sequence encoding a membrane-bound biotin mimetic peptide (BMP) or biotin acceptor peptide (BAP). The invention also relates to a method for selection of high producer cells secreting a protein of interest.

1 . A nucleic acid molecule comprising:
 (a) a first nucleic acid sequence encoding a signal peptide, linked at its C-terminal to   (b) a second nucleic acid sequence encoding a biotin mimetic peptide (BMP) or a biotin acceptor peptide (BAP), linked at its C-terminal to   (c) a third nucleic acid sequence encoding a polypeptide stretch that allows the anchorage of the BMP or BAP to a cell membrane.   
     
     
         2 . The nucleic acid molecule according to  claim 1 , wherein the first and second nucleic acid sequences) and/or the second and third nucleic acid sequences are linked by a nucleic acid sequence encoding a linker peptide. 
     
     
         3 . The nucleic acid molecule according to  claim 1 , wherein the polypeptide stretch that allows the anchorage of the BMP or BAP to a cell membrane is CD59a of SEQ ID NO: 9. 
     
     
         4 . The nucleic acid molecule according to  claim 1 , wherein the polypeptide stretch that allows the anchorage of the BMP or BAP to a cell membrane is the synthetic carrier peptide of the amino acid sequence of SEQ ID NO: 1 directly linked to CD48-GPI. 
     
     
         5 . The nucleic acid molecule according to  claim 1 , wherein the polypeptide stretch that allows the anchorage of the BMP or BAP to a cell membrane is the synthetic carrier peptide of the SEQ ID NO: 1 linked to a transmembrane peptide. 
     
     
         6 . The nucleic acid molecule according to  claim 5 , wherein the transmembrane peptide is IGF-1R-TM of ID SEQ NO: 2. 
     
     
         7 . The nucleic acid molecule according to  claim 1 , wherein the signal peptide is the signal peptide of human growth hormone or of CD59. 
     
     
         8 . The nucleic acid molecule according to  claim 1 , wherein the polypeptide stretch that allows the anchorage of the BMP or BAP to a cell membrane is linked at its C-terminal to a polypeptide conferring selection resistance. 
     
     
         9 . The nucleic acid molecule according to  claim 8 , wherein the polypeptide conferring selection resistance is selected from the group consisting of an antibiotic-hydrolyzing enzyme, an antibiotic-scavenging protein, a metabolic enzyme and an antibiotic modifying enzyme. 
     
     
         10 . The nucleic acid molecule according to  claim 8 , wherein the polypeptide stretch that allows the anchorage of the BMP or BAP to a cell membrane is linked to polypeptide conferring-selection resistance via a linker peptide. 
     
     
         11 . The nucleic acid molecule according to  claim 9 , wherein the antibiotic-hydrolyzing enzyme is selected from the group consisting of a puromycin-hydrolyzing enzyme, such as puromycin N-acetyl transferase (PAC) and an aminoglycoside hydrolyzing enzyme, such as aminoglycoside 3′-phosphotransferase; the antibiotic-scavenging protein is an Sh ble gene product; and the antibiotics modifying enzyme is a hygromycin kinase. 
     
     
         12 . The nucleic acid molecule according to  claim 2 , wherein the linker peptide consists of the amino acid sequence of isoleucine and proline or it consists of an amino acid sequence of SEQ ID NO: 4. 
     
     
         13 . The nucleic acid molecule according to  claim 1 , which is operably linked to a promoter capable of driving the transcription of the nucleic acid molecule, and is optionally operably linked upstream to an EMCV IRES sequence. 
     
     
         14 . The nucleic acid molecule according to  claim 13 , wherein the promoter is hCMV. 
     
     
         15 . The nucleic acid molecule according to  claim 1 , wherein the second nucleic acid sequence codes for a BMP. 
     
     
         16 . The nucleic acid molecule according to  claim 15 , wherein the BMP comprises the amino acid sequence of SEQ ID NO: 5 
     
     
         17 . The nucleic acid molecule according to  claim 15 , wherein the nucleic acid molecule further comprises nucleic acid sequences encoding the synthetic carrier peptide of the amino acid sequence of SEQ ID NO: 1 directly linked to the transmembrane peptide of the amino acid sequence of SEQ ID NO: 2, the signal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6, and PAC of the amino acid sequence of SEQ ID NO:  7 . 
     
     
         18 . The nucleic acid molecule according to  claim 17 , comprising the nucleic acid sequence of SEQ ID NO: 8. 
     
     
         19 . The nucleic acid molecule according to  claim 15 , wherein the nucleic acid molecule further comprises nucleic acid sequences encoding mouse CD59a of the amino acid sequence of SEQ ID NO: 9, and the signal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6. 
     
     
         20 . The nucleic acid molecule according to  claim 18 , wherein the nucleic acid sequence is of SEQ ID NO: 10. 
     
     
         21 . The nucleic acid molecule according to  claim 14 , wherein the nucleic acid molecule further comprises nucleic acid sequences encoding the synthetic carrier peptide of the amino acid sequence of SEQ ID NO: 1 directly linked to the transmembrane peptide of the amino acid sequence of SEQ ID NO: 2, and the signal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6. 
     
     
         22 . The nucleic acid molecule according to  claim 20 , wherein the nucleic acid sequence is of SEQ ID NO: 11. 
     
     
         23 . The nucleic acid molecule according to  claim 14 , wherein the nucleic acid molecule further comprises nucleic acid sequences encoding the synthetic carrier peptide of the amino acid sequence of SEQ ID NO: 1 directly linked to the CD48-GPI anchor peptide of the amino acid sequence of SEQ ID NO: 3, and the signal peptide of CD59a of the amino acid sequence of SEQ ID NO: 6. 
     
     
         24 . The nucleic acid molecule according to  claim 22 , wherein the nucleic acid sequence is of SEQ ID NO: 12. 
     
     
         25 . The nucleic acid molecule according to  claim 1 , further comprising:
 a POI-encoding nucleic acid sequence.   
     
     
         26 . The nucleic acid molecule according to  claim 25 , wherein the POI-encoding nucleic acid sequence is operably linked upstream to an EMCV IRES sequence or downstream to a promoter capable of driving the transcription of the nucleic acid molecule, or both. 
     
     
         27 . The nucleic acid molecule according to  claim 25 , wherein the POI-encoding nucleic acid sequence is a POI-encoding nucleic acid sequence coding for a single polypeptide. 
     
     
         28 . The nucleic acid molecule according to  claim 25 , wherein the POI-encoding nucleic acid sequence comprises two POI-encoding nucleic acid sequences coding for different subunits of a POI. 
     
     
         29 . The nucleic acid molecule according to  claim 28 , wherein the subunits are the light and heavy chains of an antibody. 
     
     
         30 . The nucleic acid molecule according to  claim 25 , wherein the protein of interest is linked at its amino terminus, optionally via a linker peptide, to a signal peptide. 
     
     
         31 .- 42 . (canceled) 
     
     
         43 . The nucleic acid molecule according to  claim 26 , wherein the POI is selected from the group consisting of an Fc-fusion product, an antibody, a cytokine, a hormone, a growth factor, a neurotransmitter, an enzyme, a receptor ligand, a sialomucin, a nuclear protein, a regulatory protein and a toxin, or a functional fraction thereof. 
     
     
         44 . A vector comprising the nucleic acid molecule according to  claim 1 . 
     
     
         45 . A method for the selection of eukaryotic cells secreting a protein of interest (POI), comprising identifying cells presenting BMP on their cell surface, wherein the level of BMP presentation on the cell surface is correlated with the amount of POI secreted, the method comprising the steps of:
 (d) transfecting cells with a vector according to  claim 44 , in which the second nucleic acid sequence codes for BMP and a second vector comprising aPOI-encoding nucleic acid sequence; or with a vector comprising the nucleic acid molecule further comprising a POI-encoding nucleic acid sequence, in which the second nucleic acid sequence codes for BMP,   thereby establishing a stable pool of BMP transfected cells;   (e) labeling the BMP transfected cells with a detectable biotin-binding moiety; and   (f) identifying and isolating transfected cells labeled with the detectable biotin-binding moiety.   
     
     
         46 . A method for the selection of eukaryotic cells secreting a protein of interest (POI), comprising identifying cells presenting biotin on their cell surface, wherein the level of biotin presentation on the cell surface is correlated with the amount of POI secreted, the method comprising the steps of:
 (a) transfecting cells with a vector according to claim  32 
 wherein the second nucleic acid sequence codes for BAP and a second vector comprising a POI-encoding nucleic acid sequence; or 
 a vector comprising the nucleic acid molecule further comprising a POI-encoding nucleic acid sequence, wherein the second nucleic acid sequence codes for BAP, 
 thereby establishing a stable pool of BAP transfected cells; 
   (b) adding BirA and biotin,   (c) labeling the BAP transfected cells with a biotin-binding moiety; and   (d) identifying and isolating transfected cells labeled with the detectable biotin-binding moiety.   
     
     
         47 . The method according to  claim 46 , further comprising propagating the isolated cells and repeating steps (b) to (d). 
     
     
         48 . The method according to  claim 47 , further comprising isolating a single cell labeled with the detectable biotin binding moiety and propagating the cell to form a clone. 
     
     
         49 . The method according to  claim 48 , further comprising detecting and quantifying the secreted protein of interest, thereby selecting eukaryotic cells secreting a protein of interest. 
     
     
         50 . The method according to  claims 46 , wherein said cells present BMP on their cell surface. 
     
     
         51 . The method according to  claim 46 , wherein said cells are eukaryotic cells selected from mammalian, plant, insect and yeast cells. 
     
     
         52 . The method according to  claim 51 , wherein the mammalian cells are selected from Chinese Hamster Ovary (CHO) cells, baby mouse myeloma NS0 cells, hamster kidney (BHK) cells, human embryo kidney (HEK) cells, human retinal cells, COS cells, SP2/0 cells, WI38 cells, MRCS cells and Per.C6 cells. 
     
     
         53 . The method according to  claim 46 , wherein the cells are labeled by contacting them with a detectable biotin-binding moiety selected from the group consisting of fluorescent avidin and fluorescent streptavidin. 
     
     
         54 . The method according to  claim 46 , wherein the labeled cells are identified and isolated by the means of a FACS or magnet beads. 
     
     
         55 . The method according to  claim 53 , wherein the selected eukaryotic cells are presenting higher amounts of BMP on their surface and are secreting larger amounts of protein of interest than the transfected cells of the stable pool. 
     
     
         56 . The method according to  claim 55 , wherein the amount of BMP on the surface of the selected eukaryotic cells and the amount of protein of interest secreted by the selected eukaryotic cells are larger by at least a factor selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and up to 30-fold higher than the transfected cells of the stable pool.