Production of recombinant proteins in Chinese hamster ovary cells overexpressing the subtilisin-like proprotein converting enzyme furin.
The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin-like serine endoproteases which possess cleavage specificity for sites involving multiple basic amino acid residues and are involved in the processing of precursor proteins of a variety of regulatory peptides and proteins. One of the limiting steps in the engineering of mammalian cells designed for the overproduction of secreted proteins is the endoproteolytic cleavage of the precursor molecule to its mature biologically active form. The extremely low level of endogenous furin is likely the reason why cells are not able to fully mature overexpressed precursor proteins to their mature form. Here, we report a CHO-derived cell line genetically engineered for the production of high levels of recombinant proteins that need such endoproteolytic maturation. First, the human furin cDNA under the control of the cytomegalovirus early promoter and enhancer was introduced and overexpressed in a DHFR-deficient CHO cell line. A permanent cell line CHO-D3-FUR was established that expressed biologically active furin. Subsequently, to demonstrate the capacity of CHO-D3-FUR cells to produce recombinant proteins in a fully matured form, two derivative cell lines were established that overexpressed the von Willebrand factor (vWF) and transforming growth factor beta 1 (TGF beta 1); CHO-D3-vWF and CHO-D3-TGF beta 1, respectively. Both derivative cell lines were able to produce relatively high levels of recombinant protein in a fully matured and biologically active form. Our results illustrate the potential of the CHO-D3-FUR cell line in the production of recombinant secretory proteins that need endoproteolytic activation at the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg.
Ayoubi
T A
TA
Laboratory for Molecular Oncology, University of Leuven, Belgium.
Meulemans
S M
SM
Roebroek
A J
AJ
Van de Ven
W J
WJ
D000818:Animals; D016466:CHO Cells / Q000502:physiology*; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D045683:Furin; D006801:Humans; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000096:biosynthesis; D014841:von Willebrand Factor / Q000096:biosynthesis
D004167:Diphtheria Toxin; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014841:von Willebrand Factor; D013381:Subtilisins; D045683:Furin