Strategies for recombinant Furin employment in a biotechnological process:complete target protein precursor cleavage

Coagulation factors, amongst many other proteins, often require posttransla tional endoproteolytic processing for maturation. Upon high yield expressio n of recombinant forms of these proteins, processing frequently becomes sev erely limiting, resulting in a hampered function of the protein. In this re port, the human endoprotease Furin was used to achieve complete propeptide removal from recombinant von Willebrand Factor (rvWF) precursors in CHO cel ls. At expression beyond 200 ng rvWF/10(6) cells x day, processing became i nsufficient. Stable co- and overexpression of full length Furin resulted in complete precursor cleavage in cell clones expressing 2 mu g rvWF/10(6) ce lls x day. Rather than occuring intracellularly, processing was found to be mediated by a naturally secreted form of rFurin, present in 100 fold highe r 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 m u g rvWF/106 cells x day, failed. Truncation of the trans-membrane domain r esulted in immediate secretion of rFurin and approximately 10 fold higher c oncentrations in the conditioned medium. In cases where these high rFurin c oncentrations are not sufficient to ensure complete processing, an in vitro downstream processing procedure has to be established. Secreted affinity e pitope-tagged rFurin derivatives were constructed, the fate of which, at ex pression, was dependent on the size of the C-terminal truncation and the ty pe of the heterologous epitope added. A suitable candidate was purified by a one step affinity procedure, and successfully used for in vitro processin g. This allows complete proteolytic processing of large amounts of precurso r molecules by comparably small quantities of rFurin. Complete precursor cl eavage of a target protein at expression rates of up to approximately 200 n g, 2 mu g, and 20 mu g, as well as beyond 20 mu g/10(6) cells x day can thu s be anticipated to be accomplished by endogenous Furin, additional express ion of full length rFurin, co- expression of truncated and hence secreted r Furin, and a protein-chemical in vitro procedure, respectively.