title: Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form.
abstract: We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrPSc) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrPCsource, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.
authors.affiliation: a Influenza and Prion Disease Research Center, National Institute of Animal Health , Tsukuba , Ibaraki , Japan.
authors.forename: Morikazu
authors.initials: M
authors.lastname: Imamura
authors.affiliation: a Influenza and Prion Disease Research Center, National Institute of Animal Health , Tsukuba , Ibaraki , Japan.
authors.forename: Nobuko
authors.initials: N
authors.lastname: Kato
authors.affiliation: a Influenza and Prion Disease Research Center, National Institute of Animal Health , Tsukuba , Ibaraki , Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Iwamaru
authors.affiliation: a Influenza and Prion Disease Research Center, National Institute of Animal Health , Tsukuba , Ibaraki , Japan.
authors.forename: Shirou
authors.initials: S
authors.lastname: Mohri
authors.affiliation: a Influenza and Prion Disease Research Center, National Institute of Animal Health , Tsukuba , Ibaraki , Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
authors.affiliation: a Influenza and Prion Disease Research Center, National Institute of Animal Health , Tsukuba , Ibaraki , Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Murayama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D000939:Epitopes / Q000378:metabolism; D051379:Mice; D000072002:Prion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera; D014774:Virulence
chemical_list: D000939:Epitopes; D000072002:Prion Proteins; D011994:Recombinant Proteins
keywords: Affinity tag removal; baculovirus-derived recombinant prion protein; polyhistidine-tag; profinity eXact tag; protein misfolding cyclic amplification