High Yield Expression of Recombinant Plasma Factors: Use of Recombinant Endoprotease Derivatives In Vivo and In Vitro

Abstract

Posttranslational proteolytic processing steps are often required to render proteins mature and functionally active. However, upon high yield expression of recombinant forms of these proteins, proteolytic processing becomes severely limiting.

In this report, the mammalian endoprotease Furin was used in order to achieve complete processing of recombinant von Willebrand Factor and Factor X precursors. Significant processing of rvWF was found to be mediated primarily by naturally secreted ‘shed’rFurin accumulating in the cell culture supernatant, rather than intracellularly, upon coexpression. Coamplification of rvWF/rFurin resulted in increases in rvWF yield but, at best, maintenance of rFurin expression, implying that rFurin becomes lethal to its host cell upon overexpression. In order to establish a system allowing for the processing of large quantities of recombinant protein precursor molecules with comparably small amounts of rFurin molecules in vitro, C-terminally truncated, epitope-tagged rFurin derivatives were constructed. The fate of these molecules upon expression was investigated and suitable candidates were purified to homogeneity by a one step procedure. These molecules were used for in vitro processing in solution, in reactions consisting solely of defined components, and allowing for recycling and re-use of the rFurin. Initial attempts towards the development of a downstream processing system employing matrix bound rFurin indicate that complete proteolytic processing of largeamounts of precursor molecules by small quantities of rFurin is feasible.