This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ye, K. Articles by Ibeh, J. Search for Related Content PubMed PubMed Citation Articles by Ye, K. Articles by Ibeh, J.

 Previous Article  |  Next Article 

Journal of Virology, September 2004, p. 9820-9827, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9820-9827.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

Kaiming Ye,^1* Sha Jin,² Mohammad M. Ataai,³ Jerome S. Schultz,^1, and Jeanette Ibeh³

Center for Biotechnology and Bioengineering, Department of Bioengineering,¹ Department of Molecular Genetics and Biochemistry, School of Medicine,² Chemical Engineering Department, University of Pittsburgh, Pittsburgh, Pennsylvania³

Received 2 November 2003/ Accepted 28 April 2004

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His₆ peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His₆ peptide. Display of the His₆ tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His₆-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 µg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.

---

* Corresponding author. Mailing address: Center for Biotechnology and Bioengineering, Department of Bioengineering, University of Pittsburgh, 300 Technology Dr., Suite 410, Pittsburgh, PA 15260. Phone: (412) 383-7132. Fax: (412) 383-9710. E-mail: kaiming{at}pitt.edu.

Present address: College of Engineering, University of California, Riverside, CA 92521.

---

Journal of Virology, September 2004, p. 9820-9827, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9820-9827.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Yu, J. H., Schaffer, D. V. (2006). Selection of novel vesicular stomatitis virus glycoprotein variants from a Peptide insertion library for enhanced purification of retroviral and lentiviral vectors.. J. Virol. 80: 3285-3292 [Abstract] [Full Text]  
• Zhang, B., Jin, S., Jin, J., Li, F., Montelaro, R. C. (2005). A tumor necrosis factor receptor family protein serves as a cellular receptor for the macrophage-tropic equine lentivirus. Proc. Natl. Acad. Sci. USA 102: 9918-9923 [Abstract] [Full Text]