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Journal of Virology, December 2002, p. 12023-12031, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.12023-12031.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production

Huang-Ge Zhang,1,2 Jinfu Xie,1 Igor Dmitriev,3 Elena Kashentseva,3 David T. Curiel,3 Hui-Chen Hsu,1 and John D. Mountz1,2*

Division of Clinical Immunology and Rheumatology, Department of Medicine,1 Program for Human Gene Therapy, The University of Alabama at Birmingham, Birmingham, Alabama 35294,3 Veterans Administration Medical Center, Birmingham, Alabama 352332

Received 12 July 2002/ Accepted 14 August 2002

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

* Corresponding author. Mailing address: University of Alabama at Birmingham, 701 S. 19th St., LHRB 473, Birmingham, AL 35294-0007. Phone: (205) 934-8909. Fax: (205) 975-6648. E-mail: John.Mountz{at}ccc.uab.edu.

Journal of Virology, December 2002, p. 12023-12031, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.12023-12031.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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