Protein production

Abstract

The invention concerns the field of protein production and cell culture technology. CERT is identified as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT towards its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. The present invention shows that CERT in turn is critical for PKD activation and PKD dependent protein cargo transport to the plasma membrane. The interdependence of PKD and CERT is thus a key to the maintenance of Golgi membrane integrity and secretory transport.

1 - 38 . (canceled) 
     
     
         39 - 57 . (canceled) 
     
     
         58 . An expression vector comprising two polynucleotides, wherein:
 a. a first polynucleotide encoding for a protein having an amino acid sequence comprising a steroidogenic acute regulatory related lipid transfer (START) domain, or a derivative or mutant thereof; and   b. a second polynucleotide encoding for a protein of interest.   
     
     
         59 . The expression vector of  claim 58 , whereby the START domain protein is a mammalian START domain family member is selected from PCTP, StarD7, GPBP, StarD10, StarD8, StarD13, DLC-1, StarD4, StarD6, StarD5, MLN64, StAR, THEA-2, CACH and StarD9, or a derivative or mutant thereof. 
     
     
         60 . The expression vector of  claim 58 , whereby the START domain protein is ceramide transfer protein CERT (SEQ ID NO. 11 or SEQ ID NO. 13) or a derivative or mutant thereof. 
     
     
         61 . The expression vector of  claim 60 , whereby the mutated CERT is CERT S132A  (SEQ ID NO. 15). 
     
     
         62 . The expression vector of  claim 58 , whereby said first polynucleotide increases the protein transport in a cell via the secretory pathway. 
     
     
         63 . A cell comprising the expression vector of  claim 58 . 
     
     
         64 . The cell of  claim 63 , whereby said cell is a eukaryotic cell selected from yeast, plant, worm, insect, avian, fish, reptile and mammalian cell. 
     
     
         65 . The cell of  claim 64 , whereby said eukaryotic cell is a mammalian cell. 
     
     
         66 . The cell of  claim 65 , whereby said mammalian cell is a Chinese Hamster Ovary (CHO), monkey kidney CV1, monkey kidney COS, human lens epitheliaim PER.C6™, human embryonic kidney, HEK 293, baby hamster kidney, African green monkey kidney, human cervical carcinoma, canine kidney, buffalo rat liver, human lung, human liver, mouse mammary tumor or myeloma cell, a dog, pig or macaque cell, rat, rabbit, cat, or goat. 
     
     
         67 . The cell of  claim 66 , whereby said CHO cell is CHO wild type, CHO K1, CHO DG44, CHO DUKX-B11, or CHO Pro-5. 
     
     
         68 - 75 . (canceled)