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Expression and Purification of Mammalian PCSK9 and Characterization of its Prodomain for Extracellular Trafficking.
Author(s) -
Zhong Xiaotian,
CameronLandis Lora,
Stochaj Wayne,
Awad Tony,
Pocas Jennifer,
Stahl Mark,
Kriz Ron
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb259
Subject(s) - pcsk9 , kexin , proprotein convertase , proprotein convertases , ldl receptor , subtilisin , protein precursor , protease , biochemistry , microbiology and biotechnology , chemistry , enzyme , biology , cholesterol , lipoprotein
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted member of the proprotein convertase (PC) family that is responsible for the proteolytic maturation of secretory precursors to bioactive proteins and peptides involved in many physiological processes. Recent studies reveal that PCSK9 plays a critical role in cholesterol metabolism by regulating low density lipoprotein receptor (LDLR) protein levels and function, consistent with the links of its naturally occurred mutations to hypercholesterolemia and hypocholesterolemia. In this study, we have expressed rat PCSK9 in HEK293 cells to a level of 3mg/L for a structural and functional analysis. The protein was purified to homogeneity and found active in down‐regulating surface expression of LDLR. Yet the purified protein possesses no protease activity, likely due to the fact that PCSK9 contains a pro‐domain not only essential for its extracellular trafficking but also tightly associated with its catalytic groove. Taking advantage of the recent finding that the PCSK9 prodomain can work in trans , we further characterized this structural element by deleting its C‐terminal residues, inserting protease cleavage motifs to engineer a second processing event, and swapping with prodomains of other PC family members. The results indicate that the prodomain of PCSK9 can tolerate the residue insertions but is sensitive to the C‐terminal deletion, and possesses unique features different from other PC family members.

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