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Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co‐expressing recombinant furin
Author(s) -
Fischer Bernhard E.,
Schlokat Uwe,
Mitterer Artur,
Reiter Manfred,
Mundt Wolfgang,
Turecek Peter L.,
Schwarz Hans P.,
Dorner Friedrich
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01218-4
Subject(s) - recombinant dna , furin , von willebrand factor , microbiology and biotechnology , chemistry , clone (java method) , chinese hamster ovary cell , transfection , complementary dna , biochemistry , biology , enzyme , gene , receptor , platelet , immunology
Thorough analysis of multimer composition and molecular structure of recombinant von Willebrand factor (r‐vWF) produced by recombinant CHO cells demonstrated r‐vWF to be more intact and less proteolytically degraded than plasma‐derived vWF (pd‐vWF) [B. Fischer et al. (1994) FEBS Lett. 351, 345–348]. In contrast to pd‐vWF, r‐vWF preparations consisted of pro‐vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo‐ and hetero‐multimers. In order to ensure complete propeptide processing, a r‐vWF‐producing CHO cell clone was transfected with the cDNA of the human propeptide processing enzyme Furin. A r‐vWF/r‐Furin co‐expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters. r‐vWF obtained from these cells was fully processed. Analysis of r‐vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd‐vWF, but absence of satellite bands. Two‐dimensional electrophoretic analysis of both the primary dimer and the complete multimer pattern of r‐vWF showed that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd‐vWF is known to reflect proteolytic degradation, r‐vWF thus exhibits an integrity far superior compared to pd‐vWF.