Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin.
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.
D000818:Animals; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D045683:Furin; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013381:Subtilisins / Q000096:biosynthesis* / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D013381:Subtilisins; D045683:Furin