Document Detail


Production of a recombinantly expressed laminin fragment by HEK293-EBNA cells cultured in suspension in a dialysis-based bioreactor.
MedLine Citation:
PMID:  16571374     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Virginie Belin; Patricia Rousselle
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-03-15
Journal Detail:
Title:  Protein expression and purification     Volume:  48     ISSN:  1046-5928     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2006 Jul 
Date Detail:
Created Date:  2006-06-02     Completed Date:  2006-12-05     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  43-8     Citation Subset:  IM    
Affiliation:
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.
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MeSH Terms
Descriptor/Qualifier:
Bioreactors*
Cell Line
Cells, Cultured
Culture Media, Serum-Free
DNA, Complementary / metabolism
Dialysis
Electrophoresis, Polyacrylamide Gel
Epstein-Barr Virus Nuclear Antigens / genetics*,  metabolism
Genetic Vectors
Humans
Kinetics
Laminin / biosynthesis*,  genetics,  metabolism
Recombinant Fusion Proteins / biosynthesis*,  genetics
Time Factors
Chemical
Reg. No./Substance:
0/Culture Media, Serum-Free; 0/DNA, Complementary; 0/Epstein-Barr Virus Nuclear Antigens; 0/Laminin; 0/Recombinant Fusion Proteins; 170834-93-2/laminin alpha 3

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