Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.
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.
Zhang
Huang-Ge
HG
Division of Clinical Immunology and Rheumatology, Department of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Xie
Jinfu
J
Dmitriev
Igor
I
Kashentseva
Elena
E
Curiel
David T
DT
Hsu
Hui-Chen
HC
Mountz
John D
JD
D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D036022:Capsid Proteins; D004279:DNA, Viral; D011994:Recombinant Proteins; C442558:VP3 protein, Dependovirus