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Journal of Virology, April 2006, p. 3285-3292, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3285-3292.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Selection of Novel Vesicular Stomatitis Virus Glycoprotein Variants from a Peptide Insertion Library for Enhanced Purification of Retroviral and Lentiviral Vectors

Julie H. Yu and David V. Schaffer*

Department of Chemical Engineering and the Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, California

Received 22 November 2005/ Accepted 19 January 2006

The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering.

* Corresponding author. Mailing address: Department of Chemical Engineering, University of California, Berkeley, 201 Gilman Hall, Berkeley, CA 94720-1462. Phone: (510) 643-5963. Fax: (510) 642-4778. E-mail: schaffer{at}cchem.berkeley.edu.

Journal of Virology, April 2006, p. 3285-3292, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3285-3292.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yu, J. H., Schaffer, D. V. (2006). High-throughput, library-based selection of a murine leukemia virus variant to infect nondividing cells.. J. Virol. 80: 8981-8988 [Abstract] [Full Text]  


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