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Updated: Mar 25, 2026
Production and Purification of Baculovirus for Gene Therapy Application
Published on: April 9, 2018
Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to
Morikazu Imamura1, Nobuko Kato1, Yoshifumi Iwamaru1
1a Influenza and Prion Disease Research Center, National Institute of Animal Health , Tsukuba , Ibaraki , Japan.
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 PrPC source, 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.
Insights
Researchers developed a method to purify untagged baculovirus-derived recombinant prion protein (Bac-PrP). This highly pure Bac-PrP can be converted to its pathogenic form, aiding prion disease research and drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the infectious PrPSc form in vitro.
- Highly purified Bac-PrP is crucial for applications like antibody generation, drug development, and prion propagation models.
- Existing methods may leave affinity tags that interfere with prion conversion.
Purpose of the Study:
- To establish a purification method for untagged Bac-PrP under native conditions.
- To obtain highly purified Bac-PrP free from affinity tags for unimpeded in vitro conversion.
- To ensure the purified Bac-PrP retains its ability to convert to the pathogenic form.
Main Methods:
- Utilized a three-step affinity purification strategy.
- Combined exogenous double-affinity tags (polyhistidine and profinity eXact) with the PrP N-terminal octarepeat sequence for metal ion binding.
- Purified Bac-PrP under native conditions to maintain physiological activity.
Main Results:
- Achieved near-homogeneity of untagged Bac-PrP.
- Demonstrated that the purified Bac-PrP can be converted to its pathogenic form (PrPSc).
- The purification procedure is effective for producing highly pure, functional recombinant proteins.
Conclusions:
- A robust purification method for untagged Bac-PrP was successfully developed.
- The purified Bac-PrP is suitable as a substrate for in vitro prion conversion studies.
- This purification technique has broader applications for other eukaryotic recombinant proteins requiring high purity.
