Production of a recombinantly expressed laminin fragment by HEK293-EBNA cells cultured in suspension in a dialysis-based bioreactor.
Laminin 5 is a multifunctional extracellular matrix protein, which supports epithelial cell adhesion through multiple cell binding sites. For elaborate studies, a 35 kDa fragment localized at the C-terminal extremity of the molecule, the LG4/5 fragment was recombinantly expressed in mammalian HEK293-EBNA cells. As the production of the LG4/5 fragment by adherent cell monolayers was very low (<1 microg/ml), we used the commercially available small scale bioreactor system miniPERM. The HEK293-EBNA transfectants were adapted to grow in suspension in defined medium containing low level of fetal calf serum and produced the recombinant LG4/5 protein with quality consistent with that produced in conventional static cell culture conditions. Cells grew without forming aggregates in the bioreactor and the resulting HEK293-EBNA-LG4/5 cell line was suitable for unlimited passages in the bioreactor. 2.5x10(5) cells/ml were cultured for 25 days to reach the maximal cell density of 1.6x10(7) cells/ml. The quantification of protein synthesis revealed that the highest level of 2.4 mg of recombinant LG4/5 protein was harvested when 10(7) transfected cells/ml were injected in the bioreactor and allowed to grow for 2 days. The mean daily recombinant LG4/5 fragment product yield of 1.2 mg of protein per minifermenter shows that cultivation of HEK293-EBNA transfectants in suspension is highly convenient for the production of recombinant laminin fragments.
IFR 128 BioSciences Lyon-Gerland, Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, Univ. Lyon 1, 7 passage du Vercors, 69367 Lyon, France.
Virginie
V
Belin
Patricia
P
Rousselle
D019149:Bioreactors; D002460:Cell Line; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000378:metabolism; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D007797:Laminin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors
D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D019309:Epstein-Barr Virus Nuclear Antigens; D007797:Laminin; D011993:Recombinant Fusion Proteins; C095567:laminin alpha 3