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• W1497272914 abstract "Coagulation factors, amongst many other proteins, often require posttranslational endoproteolytic processing for maturation. Upon high yield expression of recombinant forms of these proteins, processing frequently becomes severely limiting, resulting in a hampered function of the protein. In this report, the human endoprotease Furin was used to achieve complete propeptide removal from recombinant von Willebrand Factor (rvWF) precursors in CHO cells. At expression beyond 200 ng rvWF/106 cells x day, processing became insufficient. Stable co- and overexpression of full length Furin resulted in complete precursor cleavage in cell clones expressing 2 mug rvWF/106 cells x day. Rather than occuring intracellularly, processing was found to be mediated by a naturally secreted form of rFurin, present in 100 fold higher concentrations than endogenous Furin and accumulating in the cell culture supernatant. Attempts to increase rFurin yield by amplification, in order to ensure complete rvWF precursor processing at expression rates beyond 2 mug rvWF/106 cells x day, failed. Truncation of the trans-membrane domain resulted in immediate secretion of rFurin and approximately 10 fold higher concentrations in the conditioned medium. In cases where these high rFurin concentrations are not sufficient to ensure complete processing, an in vitro downstream processing procedure has to be established. Secreted affinity epitope-tagged rFurin derivatives were constructed, the fate of which, at expression, was dependent on the size of the C-terminal truncation and the type of the heterologous epitope added. A suitable candidate was purified by a one step affinity procedure, and successfully used for in vitro processing. This allows complete proteolytic processing of large amounts of precursor molecules by comparably small quantities of rFurin. Complete precursor cleavage of a target protein at expression rates of up to approximately 200 ng, 2 mug, and 20 mug, as well as beyond 20 mug/106 cells x day can thus be anticipated to be accomplished by endogenous Furin, additional expression of full length rFurin, co-expression of truncated and hence secreted rFurin, and a protein-chemical in vitro procedure, respectively." @default.
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• W1497272914 date "1999-01-01" @default.
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• W1497272914 doi "https://doi.org/10.1023/a:1008030407679" @default.
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